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

Figure 6.

Figure 6.

LRSAM1 regulates autophagy. (A) Western blot analysis of endogenous LC3B-II levels in U2OS cells transfected with vector or LRSAM1 for 24 h. CQ (25 μM) was added for the last 4 h and with or without EBSS for the last 2 h. (B) Quantification of LC3B-II levels relative to ACTB in cells treated as in (A). The average value in control vector-transfected cells (lane 1) was normalized as 1. Data are means ± SD of results from 3 experiments (*P < 0.05). (C) U2OS cells were transfected with the indicated siRNAs for 48 h, and then LRSAM1 mRNA was analyzed by RT-PCR. GAPDH was amplified as an internal control. (D) Western blot analysis of endogenous LC3B-II levels in U2OS cells transfected for 48 h with siControl or siLRSAM and treated with CQ (25 μM) for the last 4 h and/or EBSS for the last 2 h. (E) Quantification of amounts of LC3B-II relative to ACTB in cells treated as in (D). 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). (F) HeLa cells stably expressing GFP-LC3B were cotransfected for 48 h with siControl, siLRSAM1-1 or siLRSAM1-2. Levels of SQSTM1 and free GFP were analyzed by western blot. (G) Quantification of amounts of SQSTM1 protein or free GFP relative to ACTB in cells treated as in (F). The average value in siControl-transfected cells was normalized as 1 (lane 1). Data are means ± SD of results from 3 experiments (*P < 0.05).