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. 2021 Feb 20;11(9):4232–4250. doi: 10.7150/thno.49819

Figure 4.

Figure 4

SPTBN1 enhances the expression of SOCS1, which was required for the regulation of p65 by SPTBN1. (A-B) PLC/PRF/5 (A) or SNU-449 (B) cells were transiently transfected with siRNAs as indicated. Cells were then subjected to Western blot analysis. The relative intensities of SOCS1 to actin in two to three independent Western blotting were analyzed as in Figure 2A and 2B. Significance of the difference was evaluated using the Student's t test (*P < 0.05; **P < 0.01). (C-D) Liver tissues from two pairs of age- and gender-matched WT and Sptbn1+/- mice were analyzed by Western blotting (C). Liver tissues from two pairs of age- and gender-matched WT and Sptbn1+/- mice were lyzed and fractionated into cytoplasmic [C] and nuclear [N] fractions, and subcellular distribution of p65 and SOCS1 in each fraction was assessed by Western blotting (D). The relative intensities of p65 and SOCS1 were analyzed as in Figure 2A and 2B. (E) SNU-449 cells were transiently transfected as indicated for 48 h and were then analyzed by Western blot analysis. (F) SNU-449 cells were transiently transfected with control siRNA or siRNA to SOCS1 together with empty vector or SPTBN1 plasmid for 48 h. Cells were then analyzed by Western blot analysis. (G) SNU-449 cells were transiently transfected with control siRNA or siRNA to SOCS1 together with empty vector or SPTBN1 plasmid for 48 h. Cells were then treated with 10µM MG132 for 4 h before cell lysis. Cells were immunoprecipitated with antibody to p65 and the immunoprecipitates were then immunoblotted with anti-ubiquitin antibody to detect ubiquitination of endogenous p65.