Horseradish peroxidase conjugated goat anti rabbit secondary antibody

Proteins were separated on 4–15% Mini-Protean TGX precast gels (BioRad). Molecular weight markers (PageRuler^TM Prestained Protein Ladder) were from Thermo Scientific. Gels were stained with Bio-Safe Coomassie G-250 (BioRad, Solna, Sweden ) or, when used for immunoblots, transferred onto 2 µm PVDF membranes (BioRad). Membranes were blocked for 1 h in 5% non-fat powdered milk in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) followed by incubation for 1 h with primary antibody in 1% non-fat powdered milk in PBS. Membranes were then washed three times with PBST (PBS plus 0.1% Tween-20) followed by 1 h incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (BioRad) in 3% non-fat powdered milk in PBS. The membrane was then washed twice with PBST followed by one wash in PBS before being developed using a Pierce ECL immunoblotting substrate (Thermo Scientific, Stockholm, Sweden) and imaged using a ChemiDoc MP system (BioRad).

SDS-PAGE and Western blotting techniques were used to assess pulmonary GH pharmacokinetics. Rabbit polyclonal antibodies specific to each of the GHs were produced by Cedarlane (Burlington, Canada) as previously described (37 (link)). Mice were treated intratracheally with a single dose of 500 μg of each GH and then euthanized, and their lungs were harvested at the indicated time points. Lungs were homogenized in a cocktail of protease inhibitors (Roche), and pulmonary GH concentrations were quantified by Western blotting with rabbit anti-GH antibodies. Goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bio-Rad) was detected with a chemiluminescent substrate (Thermo-Fisher). The half-life of each GH was determined by densitometric analysis using ImageJ software. Band intensity at each time point was normalized to the intensity at the zero-hour time point. Half-life was determined as 50% of the relative intensity of the bands compared to the zero-hour time point.

Fifty milligrams of heart samples were homogenized in ice-cold Tris buffer (50 mmol/l, pH 8.0) containing 50 mM sodium vanadate and protease inhibitor cocktail (Sigma-Aldrich Co., Budapest, Hungary) and harvested in 2× concentrated SDS-polyacrylamide gel electrophoresis sample buffer. Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking (2 h with 3% nonfat milk in Tris-buffered saline), membranes were probed overnight at 4°C with antibodies recognizing the following antigens: mitofusin-2 (Mfn-2; 80 kDa; 1:1000; Cell Signaling #9482), optic atrophy protein-1 (OPA-1; 100–110 kDa, 1:1000; Abcam ab42364), Drp-1(Cell Signaling, #8570S) and phospho-specific Drp-1 Ser616 (Cell Signaling, #3455S) (95 kDa; 1:1000). GAPDH was used as loading control (Sigma, #105M4803, 1:1000) Membranes were washed six times for 5 min in Tris-buffered saline (pH 7.5) containing 0.2% Tween (TBST) before addition of goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3000 dilution; Bio-Rad, Budapest, Hungary). Membranes were washed six times for 5 min in TBST and the antibody—antigen complexes were visualized by means of enhanced chemiluminescence. The results of Western blots were quantified using the NIH ImageJ program.

Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:20,000), anti-Arx1 antibody (1:2,000), anti-Nsa2 antibody (1:10,000), anti-Rlp24 antibody (1:2,000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10,000), anti-Bud20 antibody (1:5000), provided by Vikram Panse, anti-Ytm1 antibody (1:100), provided by John Woolford, anti-Has1 antibody (1:10,000) provided by Patrick Linder, anti-Ebp2 antibody (1:10,000), provided by Keiko Mizuta, anti-Noc3 antibody (1:1000), provided by Herbert Tschochner, anti-Rpl3 antibody (1:5,000), provided by Jonathan Warner, anti-Rsa4 antibody (1:10,000), provided by Miguel Remacha, anti-Arc1 antibody (1:5000, raised in the Hurt lab), horseradish-peroxidase-conjugated anti-Flag antibody (1:10,000; Sigma-Aldrich, Cat# A8592, RRID:AB_439702), horseradish-peroxidase-conjugated anti-HA antibody (1:5000; Roche, Cat# 12013819001; RRID:AB_390917), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2,000; Bio-Rad, Cat# 166-2408EDU, RRID:AB_11125345), and secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2,000; Bio-Rad, Cat# STAR105P, RRID:AB_323002).