Fig. 6: RNF128-mediated ubiquitination of SRB1 promotes its recycling to the membrane via Rab11.

Plasma membrane (MEM) SRB1 refers to membrane SRB1 proteins purified using avidin from biotin-labeled membranes. Total SRB1 includes both membrane SRB1 and plasma SRB1 proteins, which are extracted from the whole lysate supernatant prior to avidin selection. A Western blotting analysis of total and MEM SRB1 in macrophages from RNF128-CKO and RNF128-WT mice with oxLDL stimulation. Sodium potassium ATPase (Na⁺/K⁺ ATPase) was used as a loading control for membrane protein (n = 4). B Laser confocal microscopy images of HeLa cells co-expressing either vector or Flag-tagged RNF128 with mCherry-tagged SRB1. Membrane SRB1 was marked by a yellow arrow (n = 4). Scale bar: 10 µm. C Western blotting analysis of indicated proteins immunoprecipitated with anti-LAMP2 or IgG antibody (n = 4). D Western blotting analysis of total and membrane proteins in HEK293T cells transfected with indicated plasmids (n = 4). E Western blotting analysis of total and membrane proteins in HEK293T cells (n = 4). F Western blotting analysis of indicated groups in macrophages immunoprecipitated with anti-Rab11 or rabbit IgG antibody (n = 4). G Western blotting analysis of indicated proteins in macrophages with Flag-RNF128 or vector overexpression immunoprecipitated with anti-Rab11 or rabbit IgG antibody (n = 4). H Confocal microscopy images of HeLa cells co-expressing either vector or Flag-tagged RNF128 with mCherry-SRB1 for 24 h. The colocalization of SRB1 and Rab11 was detected (n = 4). Scale bar: 10 µm. I Western blotting analysis of indicated proteins in macrophages immunoprecipitated with anti-Rab11 or rabbit IgG antibody (n = 4). J Quantification of SRB1 immunoprecipitated with anti-Rab11 antibody in I (upper) and total SRB1 protein of whole cell lysates in I (bottom) (n = 4). K Western blotting analysis of total and membrane proteins in macrophages incubated with DMSO, CQ, 3-MA, or leupeptin after oxLDL stimulation (n = 4). L, M Western blotting analysis of indicated groups in HEK293T cells immunoprecipitated with anti-Rab11 antibody (n = 4). The “n” represents the number of biologically independent samples. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was used for (J). ns not significant. Source data are provided as a Source Data file.