Clone c4

Cells were lysed with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl) complemented with 6.25 mM NaF, 20mM β-glycerophosphate, 1 mM DTT, and 1X Protease Inhibitor Cocktail (Complete EDTA-free tablets, Roche, Basel, Switzerland). A total of 20 μg protein was separated by SDS-PAGE gel electrophoresis using 4-15 % Mini-Protean TGX precast gels (Bio-Rad, Hercules, CA, USA), blotted to nitrocellulose membranes (Thermofisher Scientific), and probed with the following primary antibodies: anti-Six5 (1:500, Proteintech 22938-1-AP, Rosemont, IL, USA), anti-Actin B (1:5000, Clone C4, MAB1501, Millipore, Burlingotn, MA, USA), at 4°C overnight. Antibodies were diluted in 5 % BSA in TBS-T. For visualization, the membranes were incubated with IRDye800CW goat-anti-rabbit or goat-anti-mouse IgG antibodies (1:5000, LI-COR, #925-32211 and 925-32210) for 1 h at RT and then scanned with an Odyssey DLX Imaging System (LiCor). PageRuler Plus Prestained Protein Ladder (Thermofisher Scientific, #26620) was used as molecular weight marker.

Embryos were obtained from an incross of pbx3b^scm8/+;pbx4^b557/+ fish. At 48 hpf, tail tips were cut from the embryos and arrayed in 96-well plates for genotyping. The remaining portions of the embryos were arrayed in separate 96-well plates, to which sodium dodecyl sulfate (SDS) sample buffer was added and stored at −20°C until genotyping was completed, at which point the embryo lysates were pooled by genotype. Approximately one embryo equivalent was separated by reducing SDS-polyacrylamide gel electrophoresis and blotted as previously described (Maves et al., 2007 (link)). Blots were probed with anti-pan-Pbx (rabbit serum, 1:500; Pöpperl et al., 2000 (link)) and with anti-Actin as a loading control (1:500; Clone C4, Millipore). The anti-pan-Pbx antibody was raised and validated against multiple zebrafish Pbx proteins (Maves et al., 2007 (link); Pöpperl et al., 2000 (link); Waskiewicz et al., 2002 (link)). Infrared dye-labeled secondary antibodies (Rockland) were visualized using a LI-COR Odyssey infrared scanner.

About 1500–2000 synchronized worms of each strain, grown on OP50 plates at 25°C, were subjected to heat-shock (35°C for 3 h), in the first day of adulthood and were collected in M9 buffer, washed 2–3 to remove bacteria and frozen in ethanol dry ice. Control worms (no heat-shocked) of the same age were treated similarly. Likewise, ∼1500 L4-synchronized worms of each strain at 25°C, were transferred on OP50 plates containing 50 µM 5-fluoro-2'-deoxyuridine (FUDR) to prevent progeny production and collected as either 1-day or 5-day adults in M9 buffer. After 2–3 washes to remove bacteria they were frozen in ethanol dry ice. In all cases, before loading onto SDS-PAGE, worm pellets were boiled in 2× SDS-sample buffer for 10 min. Worm lysates were resolved on a 10% SDS-PAGE, western blotted and analyzed with primary antibodies against to either GFP (1∶10,000, BD Biosciences) or actin (1∶5,000, Clone C4, Millipore). Secondary anti-rabbit and anti-mouse IgG antibodies (HRP) were used respectively for immunoblot signal detection with ECL (Thermo Scientific). Quantification of immunoblot signals was performed using ImageJ software. Ratio of DCAP-1::GFP to actin levels was measured in two independent experiments.

Cells were lysed with lysis buffer (20 mM Tris-HCl (pH8.0), 1% SDS, and 1 mM DTT). Blots were probed with a mouse monoclonal antibody against BNIP3 (Clone ANa40; Abcam; ab10433), rabbit monoclonal antibody against cleaved Caspase3 (Asp175; Clone 5A1E; Cell Signaling Technology; #9664), rabbit monoclonal antibody against SAPK/JNK (Clone 56G8; Cell Signaling Technology; #9258), rabbit monoclonal antibody against phospho SAPK/JNK (T183/Y185; Clone 81E11; Cell Signaling Technology; #4668), rabbit polyclonal antibody against phospho c-Jun (S63; Cell Signaling Technology; #9261), rabbit monoclonal antibody against p44/42 MAPK (Erk1/2; Clone 137F5; Cell Signaling Technology; #4695), rabbit monoclonal antibody against phospho p44/42 MAPK (T201/Y204) (Clone D13.14.4E) (Cell Signaling Technology; #4370), rabbit polyclonal antibody against LC3B (Cell Signaling Technology; #2775), rabbit polyclonal antibody against p62 (MBL; PM045), and mouse monoclonal antibody against Actin (Clone C4) (Merck-Millipore, Billerica, MA, USA; MAB1501). Horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG secondary antibody (Cell Signaling Technology; #7076, #7074) was used as a probe, and immunoreactive bands were visualized with the Immobilon Western Chemiluminescent HRP substrate (Merck-Millipore). The band intensity was measured using ImageJ software (NIH, Bethesda, MD, USA).

Total protein extracts were obtained from 10⁶ amoebae incubated in lysis buffer (10% SDS, 10 mM Tris/HCl) containing a protease inhibitor cocktail (20 mM leupeptine (Sigma-Aldrich, USA), 50 mM N-ethylmaleimide (Sigma-Aldrich, USA), 5 mM p-chloromercuribenzoate (Sigma-Aldrich, USA), 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (Sigma-Aldrich, USA), 2 × complete mini EDTA-free (protease inhibitor cocktail, Roche, Suisse), a tablet of phosSTOP (Roche, Suisse), 10 mM E-64, 2 mM Na₃VO₄, 100 mM NaF, 10 mM iodoacetamide and 1% SDS). Crude cell extracts were boiled for 5 min at 100°C, cooled in ice for 1 min, aliquoted, and stored at −20°C. Protein samples (equivalent to 80,000 cells per lane) were resolved by 10% SDS-PAGE and electrotransferred onto a 0.2 μm PVDF membrane (Immobilon PSQ, Millipore). Proteins were detected by immunoblotting using mouse anti-actin monoclonal antibody (1:50 dilution; Clone C4, Merck Millipore, Germany), anti-HaloTag mouse monoclonal antibody (1:200 dilution, Promega, USA) or polyclonal anti-Arp3 (1:500 dilution, this work); the secondary antibodies were sheep peroxidase-conjugated anti-mouse (1:10,000; G&E) or anti-rabbit (1:20,000; G&E, USA) IgG. Membranes were treated with ECL Western blotting detection reagent and then exposed to Kodak Biomax film.