Horse anti mouse igg hrp linked

β-lactoglobulin B (βLG) from bovine milk (18.2 kDa) (#L8005) and bovine serum albumin (BSA) (66.5 kDa) (#A7906) were purchased from MilliporeSigma, and used as negative control proteins. Rabbit anti-amyloid-β (#8243), mouse-anti-amyloid precursor protein (APP) (#2452), goat anti-rabbit IgG-HRP-linked (#7074) and horse anti-mouse IgG-HRP-linked (#7076) antibodies were obtained from Cell Signaling Technology. Affinity purified anti-FAIM antibody was obtained from rabbits immunized with CYIKAVSSRKRKEGIIHTLI peptide (located near the C-terminal region of FAIM) as previously described (Kaku and Rothstein, 2009a (link),b (link)).

Goat anti-HSP27 (M-20), and mouse anti-αA-crystallin (B-2) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-vimentin, rabbit anti-histone H3, rabbit anti-MEK1/2, rabbit anti-HSP40, rabbit anti-HSP60, rabbit anti-HSP70, rabbit anti-HSP90, rabbit anti-AIF, goat anti-rabbit IgG-HRP-linked and horse anti-mouse IgG-HRP-linked antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-FLAG (M2) and mouse anti-β-actin antibodies were obtained from Millipore Sigma (St. Louis, MO, USA). Rabbit αB-crystallin antibody was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Affinity purified anti-FAIM antibody was obtained from rabbits immunized with CYIKAVSSRKRKEGIIHTLI peptide (located near the C-terminal region of FAIM) as previously described [33 (link),46 (link)].

Anti-SOCS2 (#2779), β-actin (8H10D10) (#3700), phosphorylated mTOR (p-mTOR) (Ser2448) (#5536), mTOR (7C10), rabbit monoclonal antibody (mAb) (#2983), goat antirabbit immunoglobulin G (IgG) [horseradish peroxidase (HRP) linked] (#7074), and horse antimouse IgG (HRP linked) (#7076) were purchased from the Cell Signaling Technology (Danvers, Massachusetts, United States); anti-SQSTM1/p62 antibody (ab155686), anti-ubiquitin antibody (ab7254), anti-LAMP2 antibody-lysosome marker (ab25631), recombinant anti-GSK3β antibody (Y174) (ab32391), anti-GSK3β (phospho Y216 and Y279) (ab75745), and recombinant anti-LC3B antibody (ab192890) were purchased from the Abcam (Discovery Drive Cambridge Biomedical Campus, Cambridge, United Kingdom). Antiadvanced glycation end product (AGE) carboxymethyl-lysine (CML) (MABN1837) was purchased from the Millipore (Billerica, MA, United States). Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States). The autophagy inhibitor chloroquine (CQ) diphosphate (c6628) was purchased from Sigma-Aldrich (St Louis, Mosby, United States).

The tissues were homogenized in RIPA lysis buffer (Abcam ab156034, supplemented with phosphatase inhibitor cocktail, Roche 04906845001) for protein extraction. Bradford protein assay was used to determine protein concentration with standard curves established using BSA solution. A fixed amount of the protein extract was subjected to electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated for 1 h in 5% BSA at room temperature and incubated overnight at 4 °C with the appropriate primary antibodies. Target proteins were detected by various primary antibodies (for phosphor-CREB rabbit mAb, Cell Signaling Technology #9198 (Danvers, MA, USA); for CREB rabbit mAb, Cell Signaling Technology #4820 (Danvers, MA, USA); for normalizing control β-Actin mouse mAb, Santa Cruz sc-47778 (Dallas, TX, USA)), with HRP-conjugated secondary antibodies (Goat Anti-Rabbit Immunoglobulins/HRP, Dako P0448 (Santa Clara, CA, USA); Horse Anti-Mouse IgG, HRP-linked, Cell Signaling Technology #7076 (Danvers, MA, USA)). The BCIP/NBT substrate was used for detection. The results were quantified using a Bio-Rad ChemiDoc Imaging System (Life Science, Hercules, CA, USA) and analyzed using ImageJ software.

For immunoblotting, antibodies used in this study were purchased from EMD Millipore (Oakville, Canada), Sigma Aldrich (Oakville, Canada), Proteintech (Illinois, USA), and Cell Signaling Technology (Whitby, Ontario). The following primary antibody was used from EMD Millipore (Oakville, Canada): anti-androgen receptor (Millipore Cat# 06-680, 1:1,000). The following primary antibodies were used from Sigma Aldrich (Oakville, Canada): anti-fast skeletal myosin (M4267, 1:15,000), anti-slow skeletal myosin (Sigma-Aldrich Cat# M8421, 1:10,000), and anti-beta actin (Sigma-Aldrich Cat# A2066, 1:10,000). The following primary antibodies were used from Proteintech (Illinois, USA): anti-PGC1α (Proteintech Cat# 66369-1-Ig, 1:5,000), anti-NRF-2 (Proteintech Cat# 16396-1-AP, 1:500), and anti-TFAM (Proteintech Cat# 22586-1-AP, 1:1,000). The following HRP-conjugated secondary antibodies were used from Cell Signaling Technology (Whitby, Ontario): goat anti-rabbit IgG, HRP-linked (Cell Signaling Technology Cat# 7074, 1:5,000) and horse anti-mouse IgG, HRP-linked (Cell Signaling Technology Cat# 7076, 1:5,000).