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. 2016 Aug 18;113(35):9816–9821. doi: 10.1073/pnas.1611189113

Fig. 3.

Fig. 3.

CLCa regulates TGFβR2 internalization and signaling. (A) Mean fluorescence intensity (MFI) of TGFβR2 surface labeling of follicular (Fo) and germinal center (GC) B cells from Peyer’s patches (Pp) and total B cells from spleen of WT and CLCa-null (KO) littermates (n > 11 for Pp and n = 5 for spleen, *P < 0.05, **P < 0.01; P values, unpaired t test). (B) Ratio of the MFI of WT or KO donor cells (CD45.2+) to the MFI of B6 donor cells (CD45.1+CD45.2+) for surface labeling of TGFβR2 for Fo B and GC B from WT/B6 and KO/B6 chimeras (mean ± SEM of n = 10 from at least three pairs of different donors for mixed chimera mice,**P < 0.01, ***P < 0.001; P values, unpaired t test). (C) TGFβR1 and TGFβR2 mRNA levels in spleen B cells of WT and KO littermates, normalized to mRNA levels for hypoxanthine guanine phosphoribosyl transferase 1 (mean ± SEM of n = 3). (D) Lysates (equal protein loading) of B cells from WT and KO littermates were analyzed by immunoblotting for TGFβR2 and other proteins indicated by arrowheads on the left. Migration position of molecular mass markers [kilodaltons (kDa)] is indicated. (E) HEK293T cells were transiently transfected with TGFβR2-IRES-GFP and siRNA targeting CHC17 (blue), CLCa and CLCb (red, CLCab), or scrambled siRNA (control). Percent TGFβR2 internalization at 37 °C over time was quantified after labeling with primary antibody at 4 °C and using a secondary antibody to detect residual surface receptor relative to cells maintained at 4 °C (mean ± SEM of n = 5 independent experiments, ***P < 0.001; P values, two-way ANOVA followed by Bonferroni post test). (F) Internalization of endogenous transferrin receptor (TfR) at 37 °C over time analyzed by flow cytometry for cells treated as in E (mean ± SEM of n = 5 independent experiments, ***P < 0.001; P values, two-way ANOVA followed by Bonferroni post test). (G) Levels of pSmad2/3 and Smad2/3 in lysates of spleen or B cells from WT and KO littermates quantified by immunoblotting, normalized to CHC17 levels relative to total protein loaded (representative blots in Fig. S3 E and F) (n = 5 for total splenocytes and n = 2 for purified spleen B cells, *P < 0.05; P values, one-way ANOVA).