A11122

Fat bodies (n = 4–6 pooled/sample) were sonicated in lysis buffer (2% SDS, 60 mM Tris-HCl, pH 6.8) with phosphatase and protease inhibitors (Roche). For hemolymph blots, hemolymph was collected from five larvae and pooled, and dilute hemolymph (equivalent to 50 nL of neat hemolymph) was separated by SDS-PAGE. Equal amounts of fat body or whole larval protein (10–40 μg/lane, measured using a BCA assay (Pierce)) were separated by SDS-PAGE, transferred to nitrocellulose, blocked in 3% milk in 1X TBS with 0.2% Tween 20 (TBS-T), and blotted overnight at 4°C with primary antibodies diluted in 1% milk in TBS-T. Following multiple washes in TBS-T, secondary antibodies were incubated in 1% milk in TBS-T for 2h at room temperature, washed again, incubated with ECL (Pierce), and exposed to film. Antibodies used were: rabbit anti-GFP (A11122, Invitrogen), rabbit anti-human Histone H3 and rabbit anti-HA (4499 and 3724, Cell Signaling Technology), rabbit anti-Drosophila easily shocked [78 (link)], guinea pig anti-Drosophila Pcyt1 [35 (link)], mouse anti-human SREBP1 (SC-13551, Santa Cruz Biotechnology), rabbit anti-GFP (A11122, Invitrogen), goat anti-rabbit HRP and goat anti-mouse HRP (111-035-003 and 115-035-003, Jackson ImmunoResearch), and goat anti-guinea pig HRP (6090–05, SouthernBiotech).

Primary antibodies used for immunostaining were as follows: rabbit anti-GPR157 (1:100), mouse anti-ZO1 (1:100, 33-9100; Zymed), mouse anti Ac-tub (1;500, T6793; Sigma Aldrich), rat anti-GFP (1:2000, 04404-84; Nacalai Tesque), rabbit anti-GFP (1:1000, A-11122; Invitrogen), rabbit anti-PAX6 (1:1500, PRB-278 P; Covance), rabbit anti-TBR2 (1:5000, ab23345; Abcam), rat anti-EOMES (1:4000, 14-4875; eBioscience), rabbit anti-SOX2 (1:4000, 23064; Cell Signaling Technology), goat anti-SOX2 (1:200, sc17320; Santa Cruz Biotechnology), mouse anti-TUJ1 (1:5000, MMS-435 P-250; Covance), mouse anti-S100β(1:100, S2532; Sigma Aldrich), mouse anti-DYKDDDDK (1:200, 018-22381; Wako), rat anti-BrdU (1:500, MCA2060T; Bio Rad) and mouse anti-BrdU (1:500, M0744; Dako).
Primary antibodies used for immunoblotting were as follows: rabbit anti-GPR157 (1:1000), rabbit anti-GFP (1:1000, A-11122; Thermo Fisher), and mouse anti-β-actin (1:15000, A1978; Sigma Aldrich).

Third-instar wandering larvae of each YRab line were dissected according to standard protocol to expose the dorsal tracheal system and fixed for 15-20min in 8% paraformaldehyde. Fixed filets were kept in blocking buffer (1% w/v bovine serum albumin in phosphate-buffered saline) for 45min before incubation with anti-GFP antibody A-11122 (Thermo Fisher) diluted 1:500 in blocking buffer at 4° overnight. Filets were then washed three times for 10min in PBS with 0.1% Triton-X and incubated with Alexa568-conjugated anti-rabbit secondary antibody diluted 1:500 in blocking buffer at room temperature for 1h. Filets were mounted in Vectashield medium with diamidino-phenylindole (DAPI; Vector Laboratories). We tested five different rabbit anti-GFP antibodies: A-11122 (Thermo Fisher), TP401 (Torrey Pines Biolabs), mAb anti-GFP (Upstate Biotec), Ab290 (Abcam) and antiGFP 598 (MBL). All five produced substantial unspecific staining in TCs of w- control larvae. This staining had a punctate distribution, similar to the distribution that Rabs typically show. We therefore quantified the staining intensity, comparing TCs of yrab larvae to w¹¹¹⁸ larvae that were treated in parallel with the same batch of antibody.

Frozen tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing cOmplete protease inhibitor (Roche). Protein concentrations were determined by BCA assay (Thermo Fisher Scientific). SDS-PAGE was performed using equal quantities of protein transferred to a PVDF membrane. Antibodies used at a dilution of 1 in 1,000 included anti-BSCL2/seipin, which is only capable of reacting with human BSCL2/seipin (#23846, Cell Signaling), anti-GFP (A-11122, Invitrogen), and anti-calnexin (ab75801, Abcam). Anti-rabbit HRP secondary antibody was used at a dilution of 1 in 5,000 (#7074, Cell Signaling) and visualized using enhanced chemiluminescence (Immobilon Crescendo Western HPR Substrate, Millipore, #WBLUR0500).

Whole-mount immunofluorescent staining was performed as previously reported (Ning et al., 2013 (link)). Briefly, embryos were fixed with 4% phosphate-buffered paraformaldehyde and washed with 0.3% Triton X-100 and 0.1% Tween-20 in PBS for 20 min before immunostaining. The embryos were stained with the indicated antibodies, including anti-GFP (1/1000; A-11122, Invitrogen), anti-GFP (1/1000; A-11120, Invitrogen), anti-Collagen type II (Col2) (1/100; II-116B3, Developmental Studies Hybridoma Bank), anti-pH3 (1/1000; 3377, Cell Signaling Technology) and anti-p-Smad1/5/8 (1/200; 9511, Cell Signaling Technology). All immunofluorescent images were captured using a Nikon A1R+ confocal microscope with the same settings for all experiments.
TUNEL assays were performed using the In Situ Cell Death Detection Kit, TMR red (12156792910, Roche) according to the manufacturer's instructions. DAPI was used to visualize nuclei.