Ab16771

Cells were plated on coverslips (Marienfeld precision cover glasses 1.5 H) fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.5% TritonX-100 in PBS for 5 min. In the case of digitonin treatment to decrease cytosolic staining, cells were treated with PBS containing 0.01% digitonin for 5 min on ice, followed by fixation with 4% paraformaldehyde, as described previously^{45 (link)}. Fixed cells were incubated with primary antibodies against PLIN1 (20R-pp004; Fitzgerald Industries, 1:300), ATGL (2138S; Cell Signaling, 1:300), PMP70 (ab3421; Abcam, 1:300 (all fluorescence images of immunostained PMP70 except for Supplemetary Fig. 4d), SAB4200181; Sigma, 1:300 (fluorescence images of immunostained PMP70 for Supplementary Fig. 4d), PEX5 (ab125689; Abcam, 1:300 (fluorescence images of immunostained PMP70 for Fig. 6a), sc-23188; Santacruz, 1:200 (fluorescence images of immunostained PMP70 for Supplementary Fig. 4d)), Catalase (ab16771, Abcam, 1:300) overnight, washed three times for 5 min each, incubated in secondary antibody for 1–2 h, washed three times for 5 min each, and mounted on glass slides with mounting medium (Vectashield without DAPI; Vector Laboratories). Cells were observed and imaged using an LSM 700 confocal microscope, and an OMX SIM microscope.

Cells were lysed on ice with modified RIPA buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5 mM NaF, 1 mM Na₃VO₄, and a protease inhibitor cocktail (GeneDEPOT; #P3100). Antibodies against ATGL (2138S, RRID:AB_2167955, Cell Signaling, 1:1000), HSL (4107S, RRID:AB_2296900, Cell Signaling, 1:1,000), pPKA substrate (9624S, RRID:AB_10692481, Cell Signaling, 1:1000), PLIN1 (20R-pp004, Fitzgerald Industries, 1:1000), PEX5 (NBP2-38443, Novus biological, 1:1000), CATALSE (ab16771, Abcam, 1:1000), MYC (9B11, Cell signaling, 1:1000), GAPDH (LabFrontier, Co., LF-PA0018, 1:1000), and β-actin (Sigma, A5316, RRID:AB_476743, 1:2000) were used for western blotting analysis. Original full immunoblots were shown in Supplementary Fig. 8.