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. 2022 Nov 12;298(12):102704. doi: 10.1016/j.jbc.2022.102704

Figure 3.

Figure 3

Parkin promotes K11- and K33-linked ubiquitination of PHB2 on 142/200 sites.A, FLAG vector or FLAG-PHB2 with EGFP-Parkin (WT or C431F mutant) were transfected into HEK293T cells, followed by treatment with DMSO or 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody and then were analyzed by immunoblot with indicated antibodies, n = 3. B, FLAG vector or FLAG-PHB2, HA-OPTN (WT or E478G mutant), along with mCherry-Parkin (WT or C431F mutant) were transfected into si-OPTN HEK293T cells, followed by treatment with 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody and then were analyzed by immunoblot with indicated antibodies, n = 3. C, sequence alignment of PHB2 and PHB homologs across different species. Blue shaded area indicates the lysine residues which are evolutionarily conserved. D, FLAG vector or FLAG-PHB2 (WT, K97R, K142R or K200R mutant), along with EGFP-Parkin were transfected into HEK293T cells respectively, and then the cells were treated 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody and then were analyzed by immunoblot with indicated antibodies, n = 3. E, EGFP vector or EGFP-Parkin, FLAG-PHB2 (WT or KR mutant) along with WT HA-Ub were transfected into HEK293T cells respectively, and then, the cells were treated with DMSO or 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody and then were analyzed by immunoblot with indicated antibodies, n = 3. Here and hereafter KR mutant FLAG-PHB2 indicates K142R/K200R mutant FLAG-PHB2. F, EGFP vector or EGFP-Parkin, FLAG-PHB2 (WT or KR mutant) along with HA-Ub (K6, K11, K33, K48 or K63-only) were transfected into HEK293T cells respectively, followed by treatment with 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody and then were analyzed by immunoblot with indicated antibodies, n = 3. G, quantification of co-immunoprecipitated Ub of K11 and K33 in (F), and the WT FLAG-PHB2 with GFP-Parkin expressing group were normalized as 100%. Data from three independent experiments were indicated as Means ± SD, ∗∗p < 0.01; ∗p < 0.05, One-way ANOVA followed by post hoc Tukey’s tests. H, the similar experiments (without KR mutant FLAG-PHB2) were separately performed in HA-Ub (K27 or K29-only) as (F), n = 3. EGFP, enhanced green fluorescent protein; OPTN, optineurin; PHB2, prohibitin 2.