Hrp conjugated goat anti rabbit igg

Bromopyruvate (3BP), lonidamine (LND), mitochondrial inhibitors, respiratory substrates, chemicals, LC3 rabbit polyclonal antibody, α-tubulin and β-tubulin mouse monoclonal antibodies, and goat anti-mouse FITC-conjugated IgG were from Sigma (Milan, Italy). Cyt c mouse monoclonal antibody, AIF goat polyclonal antibody, β-actin goat polyclonal antibody, p53 (FL-393) rabbit polyclonal antibody, p-p53 (Ser-315) rabbit polyclonal antibody, HK-II (C-14) goat polyclonal antibody, beclin 1 (E-8) mouse monoclonal antibody, goat anti-mouse HRP-conjugated IgG, goat anti-rabbit HRP-conjugated IgG, and rabbit anti-goat HRP-conjugated IgG were from Santa Cruz Biotechnology; VDAC1 rabbit polyclonal antibody was from Calbiochem. p-p53 (Ser-15) rabbit polyclonal, SQSTM1/p62 rabbit polyclonal, Akt rabbit polyclonal, and p-Akt (Ser-473) rabbit monoclonal antibodies were from Cell Signaling Technology. LC3 mouse monoclonal antibody was from NanoTools. Mito-tracker red CM-XRos, Alexa Fluor® 488 goat anti-rabbit IgG, and Alexa Fluor® 555 goat anti-mouse IgG were Molecular Probes products.

Brains were perfusion fixed with 4% paraformaldehyde and 30% sucrose solution before processing for histology. After being frozen, brains were sectioned coronally. Primary antibodies were applied in the following concentrations: GFAP and iNOS (1:100; Santa Cruz Biotechnology, Inc., USA). Immunohistochemistry followed the method with HRP-conjugated goat anti-rabbit IgG (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and then were visualized with 3, 30-diaminobenzidine (DAB, Sigma, St. Louis, MO, USA). Sections were then hydrated, cleared and mounted in DXP for microscopic analysis. Immunofluorescence followed with appropriate secondary antibodies (Alexa Fluor, Molecular Probes Inc.) in 1% BSA and 0.3% Triton X-100 in PBS after primary antibodies. Mounting medium was added on the slides prior to be covered with coverslips for observation by a laser scanning confocal microscope.

Retinal samples or samples of cells were sonicated in lysis buffer, namely mammalian protein extraction reagent (MPER; HyCell). Identical quantities of denatured proteins (40 μg/30 μl/well) then underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) as described previously [10 (link)]. After separation, the protein bands were transferred to a polyvinylidene difluoride membrane, which was treated for 12 h at 4 °C with the following primary antibodies, mouse monoclonal anti-β-actin antibody (AC-15; 1:2000; ab6276)/anti-HIF-1α antibody (1:200; H1alpha67-ChIP Grade; Abcam Inc.), rabbit polyclonal anti-VEGF antibody (A-20; 1:200; sc-152)/anti-PKM2 antibody (1:500; ab38237), or rabbit monoclonal anti-RBP2 antibody (ab177486; 1:1000; Abcam Inc.). The blots were next treated with relevant secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology Inc.) or goat anti-mouse IgG (1:5000; sc-2005) at 37 °C for 1 h. Dilution of primary/secondary antibodies was carried out in 5% fat-free skimmed milk. Finally, the membranes were processed using an enhanced chemiluminescent analysis system (HyCell) and exposed to an X-ray film (Fujifilm). The amount of each protein was then evaluated by scanning densitometry.

Cell lysis was performed with RIPA [33 (link)] or CHAPS lysis buffer purchased from ProteinSimple (San Jose, CA, USA) [36 (link)]. The total protein content was measured with bicinchoninic acid solution (Sigma-Aldrich, St. Louis, MO, USA) assay.
Equal amounts of proteins were analysed by immunoblotting [33 (link)]. Antibodies against β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), LC3 (#sc-16755, Santa Cruz Biotechnology, Dallas, TX, USA), OPTN (#10837-1-AP, Proteintech, Chicago, IL, USA) and phosphorylated-OPTN [21 (link)] were used. HRP-conjugated goat anti-rabbit IgG (#sc-2004) and donkey anti-goat IgG (#sc-2020) were purchased from Santa Cruz Biotechnology.

Total protein was extracted from tissue samples and cell lines using RIPA buffer containing PMSF. A BCA protein assay kit (Beyotime, Haimen, China) was used to measure total protein. Proteins were electrophoresed via SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were washed four times with TBST at room temperature and then incubated overnight at 4 °C with diluted primary antibody. Following extensive washing, the membranes were incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG, 1:3000; Santa Cruz Biotechnology, Santa Cruz, CA). Signals were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). Antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) served as an endogenous reference. Protein intensity was scanned on Typhoon PhosphorImager (GE Healthcare, Pittsburgh, PA) for fluorescent signal. Experiments were performed in triplicate.

The following primary antibodies were used: antibodies against Rictor (ab70374), MMP‐2 (ab37150) and MMP‐9 (ab76003) from Abcam (Cambridge, USA); antibodies against phospho‐AKT (S473) (#9721) and phospho‐AKT (T308) (#13038) from Cell Signaling Technology; and antibodies against AKT (AF6259), phospho‐CDK2 (Thr160) (AF3237), phospho‐Histone H3.1 (Ser10) (AF3358) and β‐actin (T0022) from Affinity Biosciences. HRP‐conjugated goat anti‐rabbit IgG and anti‐mouse IgG secondary antibodies were obtained from Santa Cruz (Dallas, TX, USA). Gelatin (G7041) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). MK‐2206, 8‐[4‐(1‐aminocyclobutyl)phenyl]‐9‐phenyl‐1,2,4‐triazolo [3,4‐f] 1, 6naphthyridin‐3(2H)‐one hydrochloride [1:1], was obtained from Selleck (Shanghai, China). 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) was purchased from Sigma‐Aldrich (St. Louis, MO).

Total protein samples were extracted from OS and OC tissues and from OS cells that were transfected with HDAC4-GFP or HDAC4 siRNA after transfection for 48 h by using cell lysis buffer (Thermo Scientific Pierce IP Lysis Buffer). The protein concentration was measured with a BCA Protein Assay Kit (Pierce). Fifty micrograms of protein per sample was used in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in Tris buffered saline with Tween 20 (TBST) at 37°C. After 1 h, the membranes were incubated with primary antibodies against HDAC4 (1:1,000, Abcam ab12172) and PCNA (1:500, Santa Cruz Biotechnology sc-25280) at 4°C overnight on a shaker. The next day, secondary antibodies were used to stain the membranes at 37°C for 1 h. The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000, Santa Cruz sc-2004) and anti-mouse IgG (1:10,000, Santa Cruz sc-2005). The detection was performed by using an enhanced chemiluminescence (ECL) kit.
The in vitro ubiquitination assays were performed as described above. When total protein samples were extracted from OS cells after transfection for 48 h, ubiquitinated PCNA was detected by SDS-PAGE and probed with primary antibodies (1:1,000, Cell Signaling Technology D5C7P).