Figure S2.
Autophagy flux analysis. (A) Western blot of control U2OS cells and Halo-ATG13, Halo-ATG2, or Halo-WIPI2 expressing U2OS cells stably coexpressing GFP-LC3B probed with an antibody against LC3B. Cell lysates were generated with CHAPS buffer split in half and treated with or without TEV protease. (B) Flow cytometry analysis of GFP-LC3B expression in control U2OS cells and Halo-ATG13, Halo-ATG2, or Halo-WIPI2 expressing U2OS cells stably coexpressing GFP-LC3B. (C) Quantification of median cellular GFP-LC3B signal intensity in Halo-ATG13, Halo-ATG2, Halo-ULK1, or Halo-WIPI2 expressing U2OS cells stably coexpressing GFP-LC3B cultured in complete media. (D) Western blot probed for LC3B of control U2OS cells, and U2OS cells expressing Halo-ATG13 or Halo-ATG2 grown in complete media, media lacking amino acids, or media lacking amino acids and FBS in the absence and presence of bafilomycin. (E) Western blot probed for LC3B of control U2OS cells, and U2OS cells expressing Halo-ULK1 or Halo-WIPI2 grown in complete media, media lacking amino acids, or media lacking amino acids and FBS in the absence and presence of bafilomycin. (F) Western blot probed for LC3B of control U2OS cells, and U2OS cells expressing Halo-ATG13, Halo-ATG2, Halo-ULK1, or Halo-WIPI2 grown in complete media, or media lacking glucose in the absence and presence of bafilomycin. (G) Quantification of the LC3B-II band intensity of the western blots shown in D and E (three biological replicates, mean and standard deviation). (H) Quantification of the LC3B-II band intensity of the western blot shown in panel F (two biological replicates, mean and standard deviation). (I) Western blot probed for GABARAP of control U2OS cells, and U2OS cells expressing Halo-ATG13, Halo-ATG2, Halo-ULK1, or Halo-WIPI2 grown in complete media, or media lacking glucose in the absence and presence of bafilomycin. Source data are available for this figure: SourceData FS2.

Autophagy flux analysis. (A) Western blot of control U2OS cells and Halo-ATG13, Halo-ATG2, or Halo-WIPI2 expressing U2OS cells stably coexpressing GFP-LC3B probed with an antibody against LC3B. Cell lysates were generated with CHAPS buffer split in half and treated with or without TEV protease. (B) Flow cytometry analysis of GFP-LC3B expression in control U2OS cells and Halo-ATG13, Halo-ATG2, or Halo-WIPI2 expressing U2OS cells stably coexpressing GFP-LC3B. (C) Quantification of median cellular GFP-LC3B signal intensity in Halo-ATG13, Halo-ATG2, Halo-ULK1, or Halo-WIPI2 expressing U2OS cells stably coexpressing GFP-LC3B cultured in complete media. (D) Western blot probed for LC3B of control U2OS cells, and U2OS cells expressing Halo-ATG13 or Halo-ATG2 grown in complete media, media lacking amino acids, or media lacking amino acids and FBS in the absence and presence of bafilomycin. (E) Western blot probed for LC3B of control U2OS cells, and U2OS cells expressing Halo-ULK1 or Halo-WIPI2 grown in complete media, media lacking amino acids, or media lacking amino acids and FBS in the absence and presence of bafilomycin. (F) Western blot probed for LC3B of control U2OS cells, and U2OS cells expressing Halo-ATG13, Halo-ATG2, Halo-ULK1, or Halo-WIPI2 grown in complete media, or media lacking glucose in the absence and presence of bafilomycin. (G) Quantification of the LC3B-II band intensity of the western blots shown in D and E (three biological replicates, mean and standard deviation). (H) Quantification of the LC3B-II band intensity of the western blot shown in panel F (two biological replicates, mean and standard deviation). (I) Western blot probed for GABARAP of control U2OS cells, and U2OS cells expressing Halo-ATG13, Halo-ATG2, Halo-ULK1, or Halo-WIPI2 grown in complete media, or media lacking glucose in the absence and presence of bafilomycin. Source data are available for this figure: SourceData FS2.

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