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. 2019 Apr 1;17(4):e2007044. doi: 10.1371/journal.pbio.2007044

Fig 8. TMEM41B shares structural homology and functional redundancy with VMP1.

Fig 8

(A) Schematic for vesicle fractionation protocol. (B) WT and indicated HEK293T knockout cells were fractionated as outlined in panel A. Fractions were resolved by SDS-PAGE followed by IB with indicated antibodies. Membrane fractions are loaded as 15X concentrate. (C) tfEmpty, tfVMP1, or tfTMEM41B expression constructs were integrated at the AAVS1 locus in WT and HEK293T knockout cells. Cell lysates were normalized by total protein prior to resolution by SDS-PAGE and IB with indicated antibodies. LC3-II levels relative to WT (tfEmpty) cells are indicated under each lane (normalized to tubulin). (D) Quantitation of LC3-II levels shown in C. LC3-II levels in each lane were normalized to tubulin. LC3-II levels in WT (Empty) cells were set to 1. Bar graphs show the mean ± SD of each sample from 3 independent experiments; p-values were determined using a student t test. *p < 0.02. (E) HEK293T cells from C were treated for 18 h with 100 nM BafA1 prior to gentle, mechanical lysis. The corresponding cell extracts were treated as indicated prior to being resolved by SDS-PAGE and analyzed by IB with indicated antibodies. NDP52 serves as a representative autophagy target; tubulin is unincorporated and serves as a control for proteolysis. Bar graphs show the mean ± SD of each sample from 3 independent experiments; p-values were determined using a student t test. *p < 0.02. Related to S7 Fig. Underlying data for all summary statistics can be found in S1 Data. AAVS1, adeno-associated virus integration site 1; ATG, autophagy-related; BafA1, Bafilomycin A1; GFP, green fluorescent protein; HEK, human embryonic kidney; IB, immunoblotting; LC3, microtubule-associated protein 1 light chain 3B; NDP52, nuclear dot protein 52; ns, not significant; RB1CC1, RB1 Inducible Coiled-Coil 1; SQSTM1, sequestosome 1; Sup, supernatant; TAX1BP1, tax1 binding protein 1; tf, tandem-fluorescent; TMEM41B, transmembrane protein 41B; VMP1, vacuole membrane protein 1; WIPI2,; WT, wild-type.