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. 2020 Oct 13;219(12):e202001031. doi: 10.1083/jcb.202001031

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© 2020 Endicott et al.

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Figure S2. — Controls for GFAP isoforms and Dendra2 reporter. (A) Displays which band of GFAP and pGFAP from NIH3T3 whole cell lysate (WCL) is the same molecular weight as the GFAP and pGFAP from purified lysosomes (Lys). (B–E) NIH3T3 cells depleted of LAMP2A by siRNA do not form more Dendra2 puncta in response to buparlisib and pictilisib treatment. For B–E, data were pooled from at least three independent experiments. In C, NC-DMSO = 68 cells, NC-Bup = 40 cells, Lamp2a-DMSO = 80 cells, and Lamp2a-Bup = 81 cells. In E, NC-DMSO = 62 cells, NC-Pic = 41 cells, Lamp2A-DMSO = 50 cells, and Lamp2A-Pic = 64 cells. (F) Control for knockdown efficiency under conditions used in B–E. (G and H) NIH3T3 cells treated with siRNA against Lamp2a show a small but significant decrease in LC3-II flux, indicating macroautophagy impairment, n = 6. NC is negative control siRNA. P values written above brackets are derived from unpaired t tests. Two-way ANOVA tables are displayed next to graphs where appropriate. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin; Bup, buparlisib; exp., exposure; Pic, pictilisib.