Pabg1

Samples were loaded on a NuPAGE 4% to 12% Bis-Tris protein gel and transferred onto an Immobilion PVDF membrane (Merk-Millipore) for 1 hour at 35 V in a wet transfer apparatus buffered with 12.5 mM Trizma-Base (Sigma), 96 mM glycine (Sigma), 10% methanol (Fisher Chemicals). The membrane was incubated in blocking solution [5% milk in PBST (PBS (pH 7.4) with 0.1% Tween-20 (VWR))] for 2 hours and probed overnight with the following primary antibodies: anti-β-tubulin (1:100, Developmental Studies Hybridoma Bank, DSHB #7, AB_2315513), anti-dMiro [75 (link)] (1:50,000), anti-Milton [76 (link)] (1:1,000, monoclonal antibody 2A108), anti-GFP (1:1,000, Chromotek, PABG1). Membranes were incubated for 1 hour with either an IRDye secondary antibody (for LI-COR Odyssey imaging) or with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by a 3-minute incubation with a chemiluminescent substrate (GE Healthcare) for ChemiDoc imaging. Secondary HRP-conjugated antibodies: anti-rabbit (1:5,000, NIF824), anti-mouse (1:5,000, NIF825), anti-guinea pig (1:5,000, SeraCare 5220–0366). LI-COR secondary antibody: IR Dye 800CW goat anti-mouse IgG (1:10,000).

For imaging, mouse monoclonal anti-VWF antibodies were acquired from DAKO (M061601-2) and rabbit polyclonal anti-ATP6V1G1 antibodies (16143-1-AP) were obtained from Proteintech. Secondary antibodies (AlexaFluor488, AlexaFluor594) were purchased from Molecular Probes. Rabbit polyclonal anti-ATP6V0d1 (Proteintech, 8274-1-AP), mouse monoclonal anti-α-tubulin clone B-5-1-2 (Sigma-Aldrich T5168), rabbit monoclonal anti-α-tubulin clone 11H10 (Cell Signaling Technology 2125), mouse monoclonal anti-RFP (Chromotek 6G6) and rabbit polyclonal anti-GFP (Chromotek PABG1) antibodies were used as primary antibodies in Western blot analysis.

The following antibodies were used in this work: guinea-pig anti-Duf extracellular (1:100) and rabbit anti-Duf extracellular (1:400; Weavers et al., 2009 (link)), rabbit anti-Pyd (1:100) and rat anti-PydEx5 (1:200; Carrasco-Rando et al., 2019 (link)), mouse anti-Fas3 [1:10; Developmental Studies Hybridoma Bank (DSHB), AB-528238], mouse anti-Peanut (1:50; DSHB, AB-528429) and mouse anti-Discs large (1:50; DSHB, AB-528203), rabbit anti-phospho-Histone H3 (Ser10) (1:100; Upstate/Sigma-Aldrich, 06-570), rabbit Cy3-anti-HRP (1:100; Jackson ImmunoResearch, AB_2340262), rabbit anti-GFP (1:300; Thermo Fisher Scientific, A-6455), rabbit anti-GFP (1:300; Chromotek, PABG1), rabbit anti-Zip (1:50; Kiehart and Feghali, 1986 (link)), rabbit anti-p-PKC (Thr410) (1:10; Santa Cruz Biotechnology, sc-12894-R), rabbit anti-PKCξ (C-20) (1:20; Santa Cruz Biotechnology, sc-216), rabbit anti-Patj (1:50; gift from Hugo Bellen, Bhat et al., 1999 (link)), rat anti-Sns (1:100; gift from Elizabeth Chen, Bour et al., 2000 (link)), anti-cubn2 (1:200; Atienza-Manuel et al., 2021 (link)) and anti-Sdk (1:200; Astigarraga et al., 2018 (link)). The following secondary antibodies were used: Alexa Fluor 488, 555 and 647 (Thermo Fisher Scientific) and DyLight 549 and 488 goat anti-rabbit IgGs (Vector Laboratories, DI-1549 and DI-1088). All secondary antibodies were used at 1:500 dilution.

Paw inflammation was induced by s.c. injection of 20 μl of CFA in the plantar surface of the right hind paw. The control group received 20 μl of PBS in the ipsilateral paw. Animals were treated daily with lorlatinib (Sigma-Aldrich) systemically (i.g. 1 mg/kg) or vehicle for 3 days following CFA injection. Paws were collected and were fixed for 24 hours in 4% PFA (Sigma-Aldrich) followed by 24 hours treatment with 30% sucrose. Footpads were then embedded in OCT and cut at 30 μm sections onto SuperFrost slides (VWR International). Tissues were washed 3 times in PBS and then blocked for 60 minutes at RT with a PBS solution containing 5% BSA and 1% Triton-X 100. Sections were incubated overnight in PBS 3% BSA, 0.3% Triton-X 100 at 4°C with rabbit anti-GFP (1:500, Chromotek, catalog PABG1). After washing in PBS, tissues were incubated with secondary antibodies (anti-rabbit IgG conjugated to Alexa Fluor 555; Invitrogen, catalog A21428) for 1 hour at RT. Slides were washed in PBS twice and mounted with Aqua PolyMount (Polysciences Inc.). Images were acquired on a Zeiss 510 confocal microscope. Image analysis was conducted using ImageJ software.

U-2 OS cells were grown on coverslips and sensitized to laser induced DSB formation using 5-Bromo-2-deoxyuridine (B9285-50MG, Sigma-Aldrich) for 24 h. GFP-SLF2 expression vectors were transiently transfected 24 h prior and GFP-SMC5 stable expressing cells were used for micro-irradiation. Laser micro-irradiation induced DSB formation was performed as previously described^{70 (link)} with 1 h allowed for recovery. Cells were pre-extracted using CSK buffer (100 mM NaCl, 10 mM HEPES, 3 mM MgCl₂, 300 mM Sucrose, 0.25% Triton-X-100, 1 mM PMSF) prior to fixation in formalin buffer (AMPQ43182, VWR) for 15 mins at room temperature (RT).
Fixed coverslips were blocked with 5% Bovine Serum Albumin (A7906, Sigma-Aldrich) for 1 h prior to staining with anti-γ-H2AX (Ser139) (1:1000, 05-636, Merck) and anti-GFP (1:500, PABG1, Chromotek) overnight at 4 °C. After PBS washes cells were stained with Alexa Fluor secondary antibodies and 4’,6-Diamidino-2-Phenylindole (DAPI, D1306, Molecular Probes) for 30 min at RT. After further washing, coverslips were dried completely and mounted for imaging using Mowiol (81381, Sigma-Aldrich).

Flies bearing LexAop-CD8-GFP-2A-CD8-GFP on the second chromosome were recombined to the vaha mutant. CaLexA was expressed in the IPC neurons using dILP2 Gal4 in control and vaha mutant flies. Both sets of flies also carried an additional LexAop-CD2GFP reporter on the third chromosome and ILP2HF to mark the IPCs. Control and vaha mutant flies were starved overnight, transferred to regular or high fat food, brains were dissected, fixed, and stained. CaLexA driven GFP signal in the brains was enhanced by staining with rabbit anti GFP antibody (rabbit anti GFP 1:200 Chromotek pabg1).