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. 2021 Nov 8;221(1):e202103156. doi: 10.1083/jcb.202103156

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© 2021 Zheng et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 4. — MARCH5 binds and ubiquitinates PMP70. (A) IP was performed with anti-V5 antibody in HEK293T cells transfected with PMP70-V5 and MARCH5-Myc, followed by IB for V5, Myc, and β-actin. (B) HeLa cells were transfected with MARCH5-Myc plasmids for 48 h. After 10 μM MG132 treatment for 6 h, IP was performed with anti-PMP70 antibody, followed by IB for PMP70 and Myc. (C) HEK293T cells were transfected with HA-Ub, PMP70-V5, MARCH5-Myc, and MARCH5(HW)-Myc plasmids for 48 h. After 10 μM MG132 treatment for 6 h, cell lysates were immunoprecipitated with anti-V5 antibody and then immunoblotted with indicated antibodies. (D) HeLa cells were transfected with empty vector (EV), MARCH5-Myc, or catalytically inactive MARCH5-CS-Myc plasmids for 48 h. The cell lysates were immunoblotted with indicated antibodies. (E) The protein levels in D were quantified with ImageJ, then normalized to β-actin. Values are mean ± SD, n = 3 independent experiments. n.s., not significant (P > 0.05) by two-tailed Student’s t test. (F) HeLa cells were transfected with two different shRNAs targeting MARCH5. The cell lysates were immunoblotted with indicated antibodies. (G) The protein levels in F were quantified with ImageJ, then normalized to β-actin. Values are mean ± SD, n = 3 independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (P > 0.05) by two-tailed Student’s t test.