Sheep anti mouse igg

Individual cochlear epithelia were lyzed in RIPA lysis buffer (Sigma, no. R0278) supplemented with Roche Protease Inhibitor (Sigma, no. 11697498001) and Phosphatase Inhibitor Cocktail no.2 (Sigma, no. P5726) and no.3 (Sigma, no. P0044). Following manufacture recommendations, equal amounts of cochlear protein extract were resolved on NuPAGE 4–12% Bis-Tris Gels (Invitrogen, no. NP0322BOX) and transferred to Immun-Blot PVDF membrane (Bio-Rad) by electrophoresis. Membranes were blocked in 5% no-fat dry milk in TBST and immunoblotted with rabbit anti-P-Smad2/3 (1:1000; Cell Signaling, no.8828 RRID: AB_2631089), P-Smad1/5/9 (1:1000; Cell Signaling, no.13820, RRID: AB_2493181) and mouse anti-β-actin (1:1000; Santa Cruz, no. SC-47778, RRID: AB_626632). HRP-conjugated secondary antibodies from Jackson Immuno Research were used at a concentration of 1:10,000 (goat anti-rabbit IgG, no. 111-035-003; sheep anti-mouse IgG no. 515-035-003). Signal was revealed using a Western Lightening Plus ECL kit (Perkin Elmer, no. NEL120E001EA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, no. 34096) according to manufacturer’s instructions.

Polyclonal AGAP2 antibody was produced in rabbits using a His-tagged N-terminal domain of AGAP2 (amino acids 131–279) as immunogen. Polyclonal antibody CT10 is an IgG-purified fraction of a rabbit antiserum against the cytoplasmic domain of FcγRIIA [34 (link)]. Actin monoclonal antibody was obtained from Sigma-Aldrich (Oakville, ON, Canada). Anti-phosphotyrosine (4G10) was from Upstate Biotechnology (Lake Placid, NY, USA). Texas Red-conjugated donkey anti-human IgG, HRP-labeled donkey anti-rabbit and sheep anti-mouse IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) and from GE Healthcare (Baie d’Urfé, QC, Canada), respectively. Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 594 donkey anti-rabbit IgG were from Invitrogen (Burlington, ON, Canada). α-tubulin (mouse 12G10) and LAMP1 (mouse UH1) antibodies were from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Calreticulin antibody was from Affinity Bioreagents (Golden, CO, USA).