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. Author manuscript; available in PMC: 2009 May 6.
Published in final edited form as: Mol Immunol. 2007 Sep 5;45(4):1035–1043. doi: 10.1016/j.molimm.2007.07.029

Figure 4.

Figure 4

Ig synthesis and secretion by LPS-stimulated PERK-deficient B-cells. Splenic B-cells were prepared from age- and sex-matched Perk+/+, Perk+/- and Perk-/- mice and cultured in the presence of LPS for 3 days. A, 2×106 viable cells were metabolically labeled with 75μCi/ml [35S]methionine and cysteine for 10 min. Ig μ and κ chains were immunoprecipitated from the cell lysates and resolved by SDS-PAGE under reducing conditions along with an equivalent amount of cell lysate (2×105 cells). Signals from the labeled Ig chains (upper and middle panels) and from labeled total proteins (lower panel) were captured and quantified by phosphorimaging. As compared to +/+ (set at 1), the mean ± SD for the μ signal: 1.04 ± 0.19 for +/-, n = 3; 0.90 ± 0.28 for -/-, n = 6; κ signal: 0.98 ± 0.14 for +/-, n = 3; 0.68 +/- 0.07 for -/-, n = 6. As compared to +/+ (set at 1), the mean ± SD for total protein synthesis was 0.94 +/- 0.16 for +/-, n = 3 and 0.89 +/- 0.08 for -/-, n = 4. B, Cell lysates were prepared and equal cell equivalents were assessed by immunoblotting for Ig μ and κ chains, the soluble ER chaperone BiP, the ER translocon component TRAPα, and actin as a loading control. C, Cells were harvested, washed and replated at 5×105 c/ml for 4 h. The amount of IgM secreted was determined by ELISA and plotted as mean ±SD (+/+ and +/-, n = 3; -/-, n = 4; p > 0.1).