Fig. 3: TRIM21 binds to NCAPH and regulates its protein expression. | Cell Death & Disease

Fig. 3: TRIM21 binds to NCAPH and regulates its protein expression.

From: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

Fig. 3

A, B HeLa and SiHa cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 1 μM) and proteasome inhibitor MG132 (10 μM). Total cell proteins were collected for western blotting at different time points (0 h, 3 h, 6 h, and 12 h). The addition of MG132 slowed the degradation rate of the NCAPH protein, suggesting the involvement of the ubiquitin proteasome pathway. C Total protein in HeLa cells was immunoprecipitated by agarose beads containing IgG or NCAPH antibodies and silver stained after gel electrophoresis. The protein bands with the most significant differences were subjected to mass spectrometry analysis. D, E Coimmunoprecipitation was used to detect the interaction between TRIM21 and NCAPH. NCAPH and TRIM21 antibodies were used as separate baits; F Immunofluorescence assay showing the colocalization of the NCAPH and TRIM21 proteins in HeLa and SiHa cells. NCAPH, 488 nm, green fluorescence; TRIM21, 594 nm, red fluorescence; nucleus, DAPI, blue fluorescence. G Western blotting analysis showed that interference with NCAPH induced no changes in TRIM21 protein levels in cervical cancer cells. H–J Western blotting showed the efficiency of interference with three sets of siRNAs targeting TRIM21 and a significant increase in NCAPH protein levels after siTRIM21 treatment in HeLa and SiHa cells. ***P < 0.001.

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