Extended Data Fig. 5: TRIM28 protects CtBP from degradation by SUMOylating CtBP.
From: Reciprocal stabilization of CtBP and TRIM28 represses autophagy to promote metastasis
(a) WB of P62 and CtBP in MDA-MB-231 cells treated by HBSS with increased time in combination with CQ treatment. (b, c) WB of CtBP1 and CtBP2 in MDA-MB-231 and MCF-7 cells with TRIM28 KD (b) for 3 d or 5 d or OE (c) for 3 d (d) qRT-PCR quantitation of CtBP1 and CtBP2 in MCF-7 and MDA-MB-231 cells with siTRIM28 up to 7 days (n = 3). (e) WB of CtBP in MDA-MB-231 cells treated with HBSS for 8 h, followed by 10 μM MG132 treatment for 8 h. (f) WB of CtBP in MDA-MB-231 cells with TRIM28 overexpression for 48 h, followed by increased GA treatment for 24 h. (g) Gluc PCA assay of TRIM28 + CtBP1 or CtBP2 (TR28 + C1, TR28 + C2) in HEK-293T cells treated by different concentrations of 2-D08 (24 h) or GA for 12 h, each dot represents the luminescence intensity per 100 μl reaction system (n = 4). (h) WB of P62 and LC3B in MDA-MB-231 cells, followed by increased GA treatment for 24 h, LC3B-II/LC3B-I ratio is also shown. (i) Co-IP analysis of CtBP by TRIM28 in MDA-MB-231 cells with Flag-TRIM28 FL OE or Flag-TRIM28 (C651A) OE for 48 h followed by MG132 treatment for 8 h. (j) IP analysis of CtBP1 WT, CtBP1 (k428r), CtBP2 WT and CtBP2 (K434R) ubiquitination in MDA-MB-231 cells upon TRIM28 FL OE for 48 h in combination with MG132 treatment for 8 h. (k) QRT-PCR measurement of SENPs after being KD for 48 h (n = 3). (l) WB of CtBP1 and CtBP2 in MCF-10A cells with siSENPs in combination with siTRIM28. Data are shown as the mean with s.d. (d,g,k); n = 3 biologically independent replicates (a-c,e,f,h-j,l). Statistical analysis was performed using a two-tailed, unpaired Student’s t-test (d,g,k); n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001.
