Article Title: GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion
Figure Lengend Snippet: GORASP2 is localized to autophagosomes and lysosomes upon starvation. ( a ) GORASP2 colocalizes with GFP-LC3 upon starvation. GFP-LC3 HeLa cells were treated with growth medium (ctr), BafA1, EBSS or EBSS and 400 nM BafA1 (E + B) for 4 h, stained for GORASP2, GOLGA2 and DNA. The bottom row shows higher magnifications of the indicated area in the above row. Scale bar: 10 µm in the upper four rows, 3 µm in the bottom row. ( b ) Quantification of ( a ) for the percentage of GORASP2 puncta that colocalized with GFP-LC3. ( c ) GORASP2 colocalizes with LC3 and LAMP2 but not EEA1 or LAMP1 upon starvation. HeLa cells were treated with EBSS and 400 nM BafA1 (E + B) for 4 h, stained for GORASP2, LC3, EEA1, LAMP1, or LAMP2 and DNA as indicated. The three rows on the right are higher magnifications of the boxed area in the three rows on the left. Scale bars: 10 µm in the left rows, 3 µm in the right rows. ( d ) Quantification of ( c ) for the percentage of GORASP2 puncta that colocalized with LC3, EEA1, LAMP1 or LAMP2. ( e ) Western blot of the proteinase K protection assay. HeLa cells were treated with growth medium (ctr) or EBSS and 400 nM BafA1 (E + B) for 4 h, then the collected PNS were equally divided into three tubes, one left untreated, one was incubated with 2.5 μg/ml protease K (PK) only, and one was treated with both PK and 1% TritonX-100 (TX-100) for 10 min.
Article Snippet: Each PNS was equally divided into three tubes, one was left untreated, one was incubated with 2.5 μg/ml Protease K (Thermo Fisher Scientific, AM2542), and the other was treated with both protease K and 1% Triton X-100 (from 20% stock) for 10 min on ice.
Techniques: Staining, Western Blot, Incubation