Odyssey sa system

Lysates from each of the esophageal cell types growing under basal conditions were resolved by SDS-PAGE (20 μg protein per lane, 12% SDS gel) as previously described (37 (link)). Western blots were probed with primary antibodies directed against ARHGDIB (ab88317, Abcam, Cambridge, UK, 1/500 overnight at 4 °C) or β-Actin (AC-15, Sigma, Dorset, UK, 1/5000, 2 h at RT) followed by secondary incubation with rabbit anti-mouse or goat anti-rabbit antibodies conjugated to either the IRDye™ 680RD or IRDye™ 800CW ((Li-Cor Biosciences, Lincoln, NE, 1/2000, 2 h at RT protected from light). Blots were imaged using the Odyssey SA system (Li-Cor Biosciences) as per manufacturer's recommendations.
For siRNA experiments, OE33 cells were transfected with vector, nontargeting scrambled sequence siRNA (siGENOME, Non-Targeting siRNA#3, Dharmacon, Lafayette, CO) or siRNA to ARHGDIB (siGENOME, SMARTPool, Dharmacon) as previously described (37 (link)). Cells were harvested 72 h after transfection and lysates resolved by Western blotting as previously.

All the Western blots in this study were acquired with a LI‐COR Odyssey SA system to detect fluorescently labeled secondary antibodies. The respective band intensities were measured using the automatic background subtraction (average top and bottom setting) of the Odyssey software. All quantifications were done across at least three independent experiments as detailed in the figure legends.

Western blotting was performed as previously described.6 Briefly 10⁷ Jurkat T cells were lysed in 200 μl Triton X‐100 buffer and 2·5 × 10⁵ to 7·5 × 10⁵ cells were resolved by SDS–PAGE under reducing conditions. Protein bands were detected by the LI‐COR Odyssey Sa system after developing with rabbit anti‐SAP antibody (clone FL‐128 Santa Cruz Biotechnology (Santa Cruz, CA)), mouse anti‐phosphotyrosine (clone PT‐66, Sigma, St Louis, MO), goat anti‐HA (biotinylated, Vector Laboratories) or anti‐β‐actin (clone AC‐15, Santa Cruz Biotechnology) followed by the appropriate secondary antibody or fluorophore‐conjugated streptavidin (IRDye 680LT‐conjugated anti‐rabbit IgG, IRDye 800 CW anti‐mouse IgG, IRDye 680LT‐conjugated anti‐mouse IgG, IRDye 800 CW Streptavidin; all LI‐COR Biosciences, Lincoln, NE). Quantification of protein expression was performed using LI‐COR odyssey sa software version 1.0.

For coimmunoprecipitation assays, cells transfected and stimulated with the appropriate ligands were lysed with IP buffer (Beyotime, P0013), after which whole cell extracts were collected and incubated with anti-GFP beads or Glutathione Sepharose 4B at 4°C for 1 hr or overnight. Then, the beads were washed 4–5 times with IP buffer, and the immunoprecipitants were eluted with 2× SDS loading buffer and then resolved by SDS-PAGE. Subsequently, the proteins were transferred to PVDF membranes (Millipore, ISEQ00010) and further incubated with the indicated primary and secondary antibodies. The images were visualized using an Odyssey Sa system (LI-COR).

Cell lysates from third instar wing imaginal discs and brains were prepared in lysis buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.1 mM EDTA, 1% Triton X-100, 0.1% SDS, 5% Glycerol, 1 mM PMSF, 1/10 tablet of Complete Mini Protease Inhibitor Cocktail) on ice. Protein samples were loaded onto a 10% polyacrylamide gel, along with Chameleon Duo Pre-stained Protein Ladder (LI-COR, P/N 928–60000) as a molecular weight ladder and run at 150 V. After SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, 162–0115, 0.45 μm) in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol) using the wet-tank method with a current density of 300 mA for 1 h. Prior to antibody incubation, membranes were blocked with 5% milk in PBS. Membrane was incubated with primary antibodies in PBS-T (1% Triton-X in 1xPBS)—rat anti-GFP (Chromotek, 3H9-100, 1:1,000) and mouse anti-α-tubulin (Sigma, T9026, 1:5,000) on a rotative plate at 4°C overnight. Primary antibodies were removed and washed in dH₂0 twice for 5 min each. Membrane was then incubated with secondary antibodies diluted in PBS-T—donkey α-mouse secondary (LI-COR, 926–68072, 1:20,000) labelled with a 700-nm IRDye and goat α-rat (LI-COR, 926–32219, 1:20,000) labelled with an 800-nm IRDye. Membrane was washed in dH₂0 twice for 5 min each before detection using the Image Studio Software on the Odyssey SA system (LI-COR).