Unless otherwise specified, the chemicals and laboratory wares used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and SPL Life Sciences (Pocheon, Korea), respectively. Oligonucleotide primers and nucleotide sequencing were both provided by Bioneer Inc. (Daejeon, Korea).

The pro-B cell line 38B9, established from BALB/c mice (d-haplotype) and with a μ heavy chain-negative and surrogate light chain-positive phenotype, was cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and 50 μM 2-mercaptoethanol at 37°C in a humidified CO₂ incubator.

MHC class II-associated protein complexes were prepared using an immunoprecipitation procedure described previously (12). In brief, cell lysates were precleared by incubating with a slurry of protein A-Sepharose 4B (GE Healthcare Life Sciences) with rotation for 30 min. The sample was then centrifuged at 5,000 g for 3 min, and the pellets were discarded. The collected supernatant was mixed and incubated with MK-D6 anti-I-A^d Ab with continuous rotation for 4 h at 4°C. A slurry of protein A-Sepharose 4B was added, and the mixture was incubated overnight at 4°C. The beads were then washed with lysis buffer before resuspension in sample reducing buffer.

The 38B9 cells that had been treated with LPS or LPS together with MK-D6 were lysed in lysis buffer. Each immunopellet was collected by immunoprecipitation and freeze-dried. Freeze-dried proteins were precipitated using the 2-dimensional (2-D) Clean-up Kit (GE Healthcare Life Sciences) before resuspension in rehydration buffer. The protein concentration was determined using the RC/DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Samples were loaded onto the strip holder, covered with an Immobiline Drystrip (GE Healthcare Life Sciences) and rehydrated passively at 20°C for 12 h. Isoelectric focusing was performed for a total of 20,000 V/h using a linear ramp protocol at room temperature. The strips were then equilibrated for 15 min each with buffer I and buffer II. The strips were loaded on top of the gels, and a 2-D run was performed at 70 V for 2 h. The gels were stained with Coomassie brilliant blue, and stained gels were analyzed using PDQuest™ 2-D analysis software (Bio-Rad).

Mass spectrometry analysis was performed as described previously. In brief, protein spots excised from the 2-D gel were dehydrated and dried in a concentrator. The gel pieces were then reduced with dithiothreitol and alkylated with iodoacetamide. After washing, gel pieces were swollen in digestion buffer containing sequencing-grade trypsin (Promega Co., Madison, WI, USA). Solutions containing peptides released into the buffer were collected as follows. Gel pieces were extracted twice with 0.1% trifluoroacetic acid in water for 20 min, and the soluble fractions were pooled together and dried. The final pellet contained most of the tryptic peptides from the digest and was analyzed by tandem mass spectrometry (MS-MS) using a Quadrupole Time-of-Flight (Q-TOF) mass spectrometer (QSTAR^® XL Mass spectrometer, Applied Biosystems/MDS Sciex, Foster City, CA, USA). Protein identification using the generated data was performed using MDS Sciex, and molecular masses and isoelectric points were calculated using the web-based ExPaSy computer molecular weight/isoelectric point tool.

Synthesized siRNA targeting hnRNP A2B1 (5′-GGA UUU GGC UUU GUA ACU U-3′) and a negative control siRNA (5′-UUC UCC GAA CGU GUC ACG UTT-3′) were obtained from Genepharma Co., Ltd. (Shanghai, China). To measure the level of cell signaling, 38B9 cells (10⁵) were transfected with an siRNA mixture consisting of 400 nM hnRNP A2B1 siRNA or negative control siRNA using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Transfected cells were incubated in a CO₂ incubator at 37°C. The cells were used for subsequent analysis at 24 h after transfection. After transfection, harvested cells were treated with 25 μg/ml LPS at 37°C for 30 min. The cells were then used for RNA or protein extraction.

To measure cytokines and cell maturation, 38B9 cells were transfected with an siRNA mixture consisting of 400 nM hnRNP A2B1 siRNA or control siRNA using Lipofectamine 3000 (Thermo Fisher Scientific). Transfected cells were incubated in a CO₂ incubator at 37°C for 12 h and then treated with 25 μg/ml LPS for 12 h. To measure cytokine levels in the culture supernatants, the culture supernatants were collected 12 h following LPS treatment. The levels of various cytokines in the collected supernatants were determined using the Th1/Th2/Th17 BD Cytometric Bead Array Kit (BD Pharmingen, Franklin Lakes, NJ, USA). Harvested cells were fixed in 2% paraformaldehyde and subjected to cytometric analysis using anti-mouse IgM Ab followed by incubation with a FITC-conjugated secondary Ab or used for quantitative real-time PCR.

The expression levels of various genes were determined using RT-PCR and quantitative real-time PCR. Total cellular RNA was isolated using the Easy-BLUE™ Total RNA Extraction Kit (Intron Biotechnology, Sungnam, Korea), and prepared RNA was converted into cDNA using a reverse transcription reaction system (Promega Co.) with a random primer or Jκ2 primer sets. PCR amplification was performed using a reaction mixture containing 10 pmol each of forward and reverse primers and cDNA (13). The PCR products were analyzed by electrophoresis on a 1.5% agarose gel. Quantitative real-time PCR was carried out in a 96-well plate using gene-specific primer sets and the Quantitect SYBR Green PCR Kit (Qiagen, Hilden, Germany) on the ABI 7500 system (Applied Biosystems/MDS Sciex). The expression level of the germline κ gene was normalized to that of actin via a relative quantitation (RQ) method using 7500 FAST software version 2.0.6 (Applied Biosystems/MDS Sciex).

The gene-specific forward and reverse primer sets used to amplify each gene were as follows: 1) hnRNP A2B1-F: 5′-CCG ATA GGC AGT CTG GAA AG-3′, hnRNP A2B1-R: 5′-CAT AAT TTC CTC CTC CAT AG-3′; 2) GAPDH-F: 5′-CCG ATG CCC CCA TGT TTG TG-3′, GAPDH-R: 5′-GGC CAT GCC AGT GAG CTT CC-3′; 3) actin-F: 5′-CGT ACC ACA GGC ATT GTG-3′, actin-R: 5′-CTC GTT GCC AAT AGT GAT GA-3′; and 4) germline Igκ-F: 5′-CCG GAT CCA CGC ATG CTT GGA GAG GGG GTT-3′, germline Igκ-R (Jκ2): 5′-CCA AGC TTT CCA GCT TGG TCC CCC CTC CGA A-3′.

For western blot analyses, cells were washed with cold phosphate-buffered saline and then lysed in mammalian protein extraction reagent (Thermo Fisher Scientific) or NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific) and a phosphatase inhibitor (Roche, Mannheim, Germany). Protein concentration was determined using a commercial BCA system (Thermo Fisher Scientific) with bovine serum albumin (BSA) as a standard. The lysates were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 3% BSA/Tris-buffered saline containing Tween 20 and incubated first with specific Abs and then with a horseradish peroxidase-conjugated secondary Ab. Finally, the blots were developed using the enhanced chemiluminescence detection system (GE Healthcare Life Sciences).

Abs against hnRNP A2B1, ERK, p38, JNK, actin, GAPDH, p50, p52, c-Rel, and RelB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Abs against Akt, phosphorylated (p)-Akt (Ser), p-Akt (Thr), 3-phosphoinositide-dependent protein kinase 1 (PDK1), p-PDK1 (Ser), p-ERK, p-p38, p-JNK/stress-activated protein kinase (SAPK), and Mcl-1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Statistical analysis was performed using Prism 5 (GraphPad Software, La Jolla, CA, USA). Data are presented as means±standard deviation of repeated experiments. The unpaired Student's t-test was used to compare 2 groups. Differences in mean values were considered significant at p<0.05.

We previously reported that LPS-mediated activation of 38B9 B cells was suppressed by crosslinking of MHC class II molecules via anti-MHC class II Ab treatment (14). To monitor the change in MHC class II molecule-associated proteins during this inhibition, MHC class II molecule-associated proteins were separated by 2-DE and analyzed using Q-TOF mass spectrometric analysis. We identified 10 candidate proteins showing enhanced expression after LPS stimulation and decreased expression after anti-MHC class II Ab treatment. Among them, we selected hnRNP A2B1, which regulates the transcription of genes involved in cell cycle progression and cell proliferation (15). Initially, we investigated whether hnRNP A2B1 is involved in MHC class II molecule-associated cell signaling (Fig. 1). The intensity of the spot corresponding to hnRNP A2B1 on the 2-DE gel suggested that LPS treatment of 38B9 cells increased the recruitment of hnRNP A2B1 to MHC class II molecules; this enhancement was inhibited by co-treatment with cognate anti-MHC class II Ab, which was confirmed at the hnRNP A2B1 transcript level (Fig. 1A). We detected similar enhanced expression of hnRNP A2B1 after LPS treatment and anti-MHC class II Ab-mediated inhibition of the enhanced hnRNP A2B1 expression in 38B9 cell lysates (Fig. 1B). Finally, we confirmed the direct interaction between hnRNP A2B1 and MHC class II molecules by immunoprecipitating MHC class II molecules. The level of MHC class II molecule-associated hnRNP A2B1 was measured in the immunoprecipitates and found to be increased by LPS treatment; the enhanced level of hnRNP A2B1 was reduced by anti-MHC class Ab treatment (Fig. 1C). Collectively, we confirmed that hnRNP A2B1 is associated with MHC class II molecules and involved in activation signaling in 38B9 B cells.

MAP kinase signaling plays an important role in various cellular processes. We previously demonstrated that ERK/p38 MAP kinases are involved in the proliferation and differentiation of 38B9 cells, especially in MHC class II molecule-mediated negative signaling (11). To confirm the involvement of hnRNP A2B1 in the regulation of MAP kinase signaling, we monitored the influence of siRNA-mediated hnRNP A2B1 inhibition on MAP kinase activation (Fig. 2). As expected, LPS treatment increased the level of hnRNP A2B1 expression in 38B9 B cells by approximately 1.69-fold relative to the control. Additionally, the increased levels of hnRNP A2B1 protein were decreased in hnRNP A2B1 siRNA-treated cells compared with control siRNA-treated cells (both LPS-treated and LPS non-treated) by approximately 0.47- and 0.28-fold, respectively. This suggested that the siRNA targeting hnRNP A2B1 efficiently inhibited hnRNP A2B1 expression. Importantly, knockdown of hnRNP A2B1 influenced phosphorylation of MAP kinases: the levels of p-ERK and p-p38 MAP kinases were increased by LPS by approximately 1.35- and 2.02-fold relative to the control, respectively, while the p-p38 level was decreased by hnRNP A2B1 siRNA by approximately 1.78-fold relative to the control. In contrast, the level of p-JNK MAP kinase was decreased by LPS by approximately 0.17-fold relative to the control but was recovered to 0.96-fold of the control level by hnRNP A2B1 siRNA treatment. Collectively, we confirmed that hnRNP A2B1, which is associated with MHC class II-mediated signaling, is involved in the regulation of MAP kinase signaling in LPS-stimulated 38B9 B cells.

NF-κB regulates the expression of various cytokine genes and genes involved in the activation, development, and survival of lymphocytes. In addition, LPS is one of the strongest stimulants of NF-κB activation in B cells and is capable of inducing the expression of various cytokines and cell differentiation (16). Because we previously confirmed that anti-MHC class II Ab regulates NF-κB activation in LPS-stimulated 38B9 cells, we monitored the influence of hnRNP A2B1 on NF-κB activation in LPS-stimulated 38B9 cells (Fig. 3A). LPS stimulation of 38B9 cells increased the level of hnRNP A2B1 in the cytoplasm, and the level of hnRNP A2B1 was decreased by siRNA treatment in both the cytoplasm and nucleus. The levels of NF-κB subfamily members c-Rel and p50 in the nucleus were increased by LPS treatment, presumably by their enhanced translocation from the cytoplasm and/or enhanced protein expression by LPS treatment. Additionally, the levels of c-Rel and p50 in both the cytoplasm and nucleus were decreased by hnRNP A2B1 siRNA. Conversely, other NF-κB family members, RelB and p52, showed enhanced translocation into the nuclear compartment after knocking down hnRNP A2B1 using siRNA in LPS-stimulated 38B9 cells. Moreover, the expression of IL-6 and tumor necrosis factor (TNF) was significantly (p<0.01) increased by LPS treatment, and the increased expression of these pro-inflammatory cytokines was inhibited by siRNA-mediated knockdown of hnRNP A2B1 (Fig. 3B), suggesting that hnRNP A2B1 is involved in pro-inflammatory cytokine expression in LPS-stimulated 38B9 cells. Although the activation of NF-κB-related proteins such as c-Rel and p50 was increased following hnRNP A2B1 siRNA treatment, knocking down hnRNP A2B1 did not show any activating effect on the expression of pro-inflammatory cytokines in non-LPS-stimulated 38B9 cells. Collectively, these results suggest that NF-κB activation in LPS-stimulated B cells is negatively regulated by hnRNP A2B1 knockdown, and that NF-κB activation by LPS stimulation in B cells is regulated to recruit hnRNP A2B1 to the cytoplasm.

The activation of phosphoinositide 3-kinase (PI3K) is required for B cell proliferation and survival. PI3K signaling is also one of the key aspects of B cell differentiation, and its activation promotes Ca²⁺ mobilization and activation of NF-κB-dependent gene transcription, as well as activation of Akt and mammalian target of rapamycin (mTOR) (4). Because PI3K/Akt activity is involved in B cell differentiation into plasma cells, we analyzed the involvement of hnRNP A2B1 in Akt signaling (Fig. 4). Interestingly, the levels of p-Akt at Thr308 and Ser473 were increased by LPS stimulation by approximately 3.59- and 6.21-fold, respectively, and were inhibited by hnRNP A2B1 siRNA by 1.89- and 3.31-fold, respectively, relative to the control cells. We next analyzed the downstream signaling molecules, including GSK3β and Mcl-1, a myeloid leukemia cell differentiation protein. Similar to p-Akt, the levels of p-GSK3β and Mcl-1 were increased by LPS treatment by approximately 2.01- and 1.68-fold, respectively, and were reduced by hnRNP A2B1 siRNA by 1.39- and 0.51-fold, respectively, relative to the control cells. Collectively, these results suggest that hnRNP A2B1 is involved in cell differentiation via regulation of Akt signaling.

LPS stimulation of B cells via TLR4 induces B cell maturation. 38B9 cells are a pro-B cell line, and the Ig gene is in the rearrangement status of D_H-J_H alleles but is not induced by V_H rearrangement (17). To analyze the influence of hnRNP A2B1 on B cell maturation, we assessed the status of Ig gene rearrangement in LPS-stimulated 38B9 cells after hnRNP A2B1 siRNA treatment (Fig. 5). When we initially measured the level of the germline Igκ light chain gene transcript by RT-PCR, the germline Igκ transcript was not detected in control non-stimulated cells but was detected in LPS-stimulated cells, and the level was reduced in hnRNP A2B1 siRNA-treated cells (Fig. 5A). We confirmed these results by quantitative real-time PCR, and RQ of the germline Igκ transcript was significantly (p<0.05) reduced by approximately 50% in hnRNP A2B1 siRNA-treated cells (Fig. 5B). We next confirmed the expression of surface IgM in 38B9 cells, because germline Igκ expression leads to B cell maturation. As expected, similar to germline Igκ transcript expression, LPS stimulation of 38B9 cells increased the surface IgM-positive cells, and the level of LPS-mediated enhanced surface IgM-positive cells was reduced in hnRNP A2B1 siRNA-treated cells (Fig. 5C). Collectively, these results suggest that hnRNP A2B1 is involved in B cell maturation and differentiation.