Anti ctbp2

Cochlear sections and spreads were incubated with 5% normal goat serum (ZSGB-BIO, Beijing, China) and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline (PBS) for 2 h at room temperature. After washing three times with PBS, the samples were incubated with primary antibody solution at 4°C overnight. After multiple washes, the samples were incubated with secondary antibodies at a ratio of 1:300 for 2 h at room temperature and protected from light. The primary antibodies used were anti-myosin-VIIa (1:300, Proteus BioSciences Inc. Ramona, CA), anti-CtBP2 (1:500, BD Biosciences, Franklin Lakes, NJ), anti-GluR2 (1:400, Millipore, Burlington, MA), anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, 1:300, Abcam, Cambridge, United Kingdom), anti-green fluorescent protein (GFP) (1:100, Santa Cruz Biotechnology, Dallas, TX), and anti-4-HNE (1:500; Abcam). The secondary antibodies used were goat anti-mouse IgG1 Alexa Fluor 568, goat anti-mouse IgG2a Alexa Fluor 488, and goat anti-rabbit IgG (H + L) Alexa Fluor 647 (1:300, Invitrogen/Molecular Probes, Eugene, OR). 4, 6-diamidino-2-phe-nylindole (DAPI) was used for the final addition of coverslips. The expression of 8-OHdG and 4HNE was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc. United States).

Protein extraction was performed in RIPA buffer supplemented with protease inhibitor cocktail. Western blot experiments were performed according to the standard procedure with the following antibodies: anti-CtBP1, anti-CtBP2 (BD Biosciences, Franklin Lakes, NJ, USA), anti-β-actin (Cell Signaling Technology, Danvers, MA, USA), and anti-vinculin (Sigma-Aldrich, St Louis, MO, USA). Anti-rabbit fluorescent secondary antibody (IRDye^® 800CW Donkey anti-Rabbit IgG, Li-Cor Biosciences, Lincoln, NE, USA) or anti-mouse fluorescent secondary antibody (IRDye^® 680RD Donkey anti-Mouse IgG, Li-Cor Biosciences Lincoln, NE, USA) were used. Membranes were imaged using a Li-Cor Odyssey Fc scanner and densitometry analysis was performed using Image Studio Lite software Version 5.2 (uncropped blots are provided in File S2).

We used the following primary antibodies for immunostaining: mouse monoclonal anti-Brn3a (Chemicon, MAB1585, 1:100), anti-Pax6 (DSHB, 1:100), anti-Ctbp2 (BD Biosciences, 612044, 1:500), anti-GFAP (Sigma, G3893, 1:400), anti-PKARIIβ (BD Biosciences, 610625, 1:500), and anti-RomI (1:500) (a gift from Dr. R. Molday, the University of British Columbia, Canada); rabbit polyclonal anti-Rhodopsin (LSL, LB-5597, 1:2500), anti-M-opsin (Millipore, AB5405, 1:500), anti-Chx10 (1:200)^{61 (link)}, anti-Pikachurin (1:500)^{62 (link)}, anti-PKCα (Sigma, P4334, 1:500), anti-Trpm1 (1:100)^{63 (link)}, and anti-Calbindin (Calbiochem, PC253L, 1:1,000); goat polyclonal anti-S-opsin (Santa Cruz, sc-14363, 1:500) and anti-Brn3b (Santa Cruz, sc-6026, 1:100); guinea pig polyclonal anti-mGluR6 (1:500)^{63 (link)}; rat monoclonal anti-GFP (Nacalai, 04404-84, 1:1,000) antibodies. We used Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, 1:500) and Alexa Fluor 488-conjugated secondary antibodies (Sigma, 1:500).

Western blotting analysis was performed following a previously described method 20 (link). Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, #R0278) containing protease inhibitor (Sigma-Aldrich, P8340). Equal amounts of total proteins were loaded and resolved by SDS-PAGE gels, followed by transferring to a PVDF membrane, blocking with 5% milk, and probing with primary antibodies. The following primary antibodies were used: anti-CtBP1 (BD Bioscience, USA, #612042), anti-CtBP1 (phospho Ser422) (GeneTex, USA, #GTX55356), anti-CtBP2 (BD Bioscience, #612044), anti-HIPK2 (Cell Signaling, USA, #5091S), anti-BIM (Abcam, China, #ab170589), anti-BIK (Abcam, #ab52182), anti-BAX (Abcam, #ab3191), anti-NOXA (Abcam, #114C307), anti-CASP3 (Sigma-Aldrich, #C9598), anti-CASP7 (Sigma-Aldrich, #C1104), anti-CASP9 (Abcam, #ab184786), anti-p300 (Sigma-Aldrich, #P2859), anti-FOXO3a (Sigma-Aldrich, #V38041), and anti-GAPDH (Thermo Fisher Scientific, #MA5-15738-BTIN). The protein signals were visualized using an ECL detection kit (Sigma-Aldrich, #GERPN2109).

Aggregates were fixed with 4% paraformaldehyde. The fixed specimens were cryoprotected with a serial treatment of 15% and 30% sucrose and embedded in tissue freezing medium. Frozen tissue blocks were sectioned into 10 or 12 μm cyrosections. For immunostaining, a 3% Goat or Horse Serum and 0.1% Triton-X100 solution was used for primary antibody incubation. An Alexa Fluor 488, 568, or 647 conjugated anti-mouse IgG or anti-goat IgG and an Alexa Fluor 568 or 647 conjugated anti-rabbit IgG (Invitrogen) were used as secondary antibodies. A DAPI counterstain was used to visualize cellular nuclei (ProLong Gold antifade reagent with DAPI, Life Technologies). Microscopy was performed on a Nikon TE2000 Inverted Microscope or an Olympus FV1000-MPE Confocal/Multiphoton Microscope.
The following antibodies were used: anti-E-cadherin (rabbit, Abcam; mouse, BD Biosciences); anti-Nanog (rabbit, Abcam); anti-Pax8 (rabbit, Abcam); anti-Pax2 (rabbit, Invitrogen; mouse, Abnova); anti-Sox2 (mouse, BD Biosciences); anti-myosin7a (rabbit, Proteus); anti-acetylated-α-Tubulin (mouse, Abcam); anti-TuJ1 (mouse, Covance); anti-Calbindin2 (mouse, Millipore); anti-Brn3A (mouse, Millipore); anti-Brn3C (mouse, Santa Cruz Biotechnology); anti-CtBP2 (mouse, BD Biosciences); anti-GFP (mouse, Santa Cruz); anti-GFP (rabbit, Abcam); anti-GFP (mouse, Life Technologies); anti-Espin (rabbit, Gift of Dr. James Bartles).

The immunoblot analysis was performed as described previously 35 (link). Briefly, tissues and cells were lysed in 1×RIPA buffer (Sigma-Aldrich, #R0278). Equal amounts of protein in each sample were loaded into a 10% SDS-PAGE gel. After transferring to a membrane and blocking with 5% milk, the proteins were probed with the following primary antibodies: anti-CtBP1 (BD Biosciences, San Jose, CA, USA, #612042), anti-CtBP2 (BD Biosciences, #612044), anti-CD31 (ThermoFisher Scientific, #PA5-16301), anti-CD55 (ThermoFisher Scientific, #PA5-82005), anti-CD68 (ThermoFisher Scientific, #MA5-13324), anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-365062), anti-Caspase-1 (Santa Cruz Biotechnology, #sc-56036), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Abcam, Cambridge, MA, USA, #ab9106), anti-p300 (Santa Cruz Biotechnology, #sc-585), anti-c-Jun (Sigma-Aldrich, #SAB4501606), anti-c-FOS (Sigma-Aldrich, #F7799), anti-p50 (ThermoFisher Scientific, #PA1-30409), anti-p65 (ThermoFisher Scientific, #14-6731-81), anti-IRF2 (Abcam, #ab3388), anti-STAT4 (Abcam, #ab68156), anti-NLRP3 (Abcam, #ab210491), anti-Il-1β (Abcam, #ab2105), anti-DNMT1 (Abcam, #ab13537), and anti-DNMT3A (Abcam, #ab2850). After probing with secondary antibodies, protein band signals were detected using a Pierce^TM ECL western blotting substrate (ThermoFisher Scientific, #32106).

Paraffin-embedded sections were deparaffinized and immersed in unmasking solution (Vector H3300; Vector Laboratories) for antigenic retrieval and heated in an autoclave (121 °C) for 5 min. Sections were then incubated with a non-specific blocking reagent (Dako) for 1 h to block any non-specific reactions, followed by overnight incubation at 4 °C with one of the following primary antibodies: anti-ERK1 (Invitrogen, 13–8600), anti-ERK2 (Cell Signaling, #9108), anti-p-ERK1/2 (Cell Signaling, #9101), anti-myosin VIIa (Proteus Biosciences, 25–6790), or anti-CtBP2 (BD Transductions, 612044) diluted in an antibody diluent (DAKO). Sections were washed thrice in PBS and incubated with a corresponding secondary antibody (Alexa Fluor 488 or 546, IgG, Invitrogen) diluted in an antibody diluent (DAKO). After rinsing in PBS, NSE was mounted onto slides with an antifade mounting medium (VECTASHIELD with DAPI; Vector Laboratories, Burlingame, CA, USA). DAPI labeling was then used to identify condensed HC nuclei. Images of immunolabeled specimens were obtained by confocal fluorescence microscopy using a Nikon C2 system (Nikon, Tokyo, Japan).