Skip to main content
. 2015 Jan 28;10(12):2158–2170. doi: 10.4161/auto.36439

Figure 7.

Figure 7.

PHF23 mediates autophagy by promoting ubiquitination and degradation of LRSAM1. (A) Western blot analysis of endogenous LC3B-II levels in U2OS cells transfected with the indicated plasmids for 24 h. CQ (25 μM) was added for the last 4 h. (B) Quantification of amounts of LC3B-II relative to ACTB in cells treated as in (A). The average value in control vector-transfected cells was normalized as 1. Data are means ± SD of results from 3 experiments (*P < 0.05). (C) Western blot analysis of endogenous LC3B-II levels in U2OS cells transfected for 48 h with different siRNAs and then treated with CQ (25 μM) for the last 4 h. (D) Quantification of LC3B-II levels relative to ACTB in cells treated as in (C). The average value in siControl-transfected cells (lane 1) was normalized as 1. Data are means ± SD of results from 3 experiments (*P < 0.05, **P < 0.01). (E) HeLa cells stably expressing GFP-LC3B were cotransfected with different siRNAs for 48 h. Levels of free GFP were analyzed by protein gel blot. (F) Quantification of free GFP levels relative to ACTB in cells treated as in (E). The average value in siControl-transfected cells was normalized as 1. Data are means ± SD of results from 3 experiments (*P < 0.05, **P < 0.01). (G) U2OS cells were cotransfected with polyQ80-luciferase (or polyQ19-luciferase) and the indicated siRNAs for 48 h. PolyQ80 /polyQ19 ratios were analyzed using the Dual Luciferase Reporter System (*P < 0.05, **P < 0.01). (H) HEK293 cells were transiently transfected with different combinations of mammalian expression vectors for FLAG-ubiquitin, PHF23, PHF23PHDΔ, and LRSAM1. After 24 h, cells were treated with 10 μM MG132 for 6 h. The cells lysates were then immunoprecipitated with an anti-LRSAM1 antibody and probed with an anti-ubiquitin antibody. (I) HEK293 cells were transiently transfected with different combinations of mammalian expression vectors for PHF23, PHF23PHDΔ, and LRSAM1 for 24 h, cells were treated with 10 μM MG132 for 6 h. The cells lysates were then probed with anti- LRSAM1 and PHF23 antibodies. ACTB was used as the loading control.