Goat anti mouse igg secondary antibody

To visualize CDC oligomers, SDS-AGE analysis was carried out as described previously [20 (link)]. Briefly, 1.0×10⁶ cells were washed then incubated with 5 µg ml⁻¹ of each toxin (or 30 µg ml⁻¹ for PLY where indicated) for 10 min on ice. Cells were washed twice with PBS and once with HEPES/NaCl buffer (20 mM HEPES at pH 7.5, 150 mM NaCl), before being resuspended in 40 µl of 0.01 % glutaraldehyde in HEPES/NaCl as a cross-linker and incubated at 37 °C for 15 min. Then 0.1M Tris (pH 8.0) was added to stop the cross-linking reaction. Tris/glycine SDS sample buffer (LC2676; Thermo Fisher) was added to the cell solution at a 1 : 1 ratio and the mixture was incubated at 95 °C for 20 min to lyse the cells.
The lysate was loaded onto a 1.5 % agarose gel. Proteins from the gel were transferred to a polyvinylidene difluoride (PVDF) membrane using semi-wet transfer, blocked in 5 % nonfat dry milk in Tris-buffered saline (TBS) plus 1 % Tween-20, and incubated with 6× His-tag monoclonal primary antibody (MA1-135; Thermo Fisher) overnight at a dilution of 1 : 750. The membrane was washed with TBS plus 1 % Tween-20, and incubated with goat anti-mouse IgG secondary antibody (31430; Thermo Fisher) for 1 h at a 1 : 10 000 dilution. The membrane was then washed with several changes of TBS plus 1 % Tween-20 for 3 h before being imaged on iBright CL1000 using Pierce ECL Western blotting substrate.

Whole‐cell lysates were obtained from RASSF1 and control shRNA transfected HEK293FT cells treated with tunicamycin (5 μg·mL⁻¹) for 5 h. The cells were harvested with RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA). Lysates were sonicated for 30 s, and the protein concentration was measured by DC protein assay (Bio‐Rad). Protein samples (30 μg each) were separated by 4–12% PAGE Gel (GenScript, Piscataway, NJ, USA) and then transferred onto PVDF membranes (Millipore‐Sigma, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk for 60 min at room temperature and incubated overnight at 4 °C with recombinant anti‐RASSF1 rabbit monoclonal antibody (1 : 500, ab126764; Abcam) or anti‐α‐Tubulin monoclonal mouse antibody (1 : 5000, T9026; Sigma). After washing, membranes were incubated for 1 h at room temperature with horse radish peroxidase (HRP) conjugated goat anti‐rabbit IgG secondary antibody (1 : 10 000; Abcam) or goat anti‐mouse IgG secondary antibody (1 : 10 000; ThermoFisher) at room temperature. The immobilized proteins were detected using the enhanced chemiluminescence reagent plus (PerkinElmer, Waltham, MA, USA). Images were obtained with ChemiDoc™ Touch Imaging System (Bio‐Rad) and analyzed with image lab.

Anti-β-actin mouse monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Flag or anti-HA mouse monoclonal antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). The goat anti-mouse IgG secondary antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Apoptosis Inducers Kit (C0005) was obtained from Beyotime (Shanghai, China).

Proteins were extracted from frozen heart tissues with K150T buffer (150 mM KCl, 50 mM Tris-HCl pH7.4, 0.125% Na deoxycholate, 0.375%Triton X-100, 0.15% NP-40, 4 mM EDTA, 50 mM NaF) and quantified by BCA protein assay (Thermo Scientific). Equal amount of proteins (15 µg) were resolved on 4–12% NuPAGE Bis-Tris gel and transferred to PVDF membrane. A Pierce Reversible Protein Stain Kit was used to detect total proteins for normalization of loading.
Primary antibodies used in immunoblotting were OXPHOS (Abcam ab110413, at 1:500), Troponin I (Cell Signaling Technology #4002, 1:1000), pSer23/24-Troponin I (Cell Signaling Technology #4004, 1:1000), pSer150-Troponin I (ThermoFisher PA5-35410; 1:1000), cMyBP-C (Santa Cruz SC-137237, 1:1000), pSer282-cMyBP-C (ALX-215–057 R050, 1:2000).
Secondary antibodies used were donkey anti-rabbit IgG secondary antibody and goat anti-mouse IgG secondary antibody (both from Thermo Scientific). SuperSignal West Pico Chemiluminescent Substrate was used for detection and AlphaView Software (Protein Simple, San Jose, CA), was used for image acquisition and quantification.

Proteins were extracted from heart tissues with K150T buffer (150 mM KCl, 50 mM Tris-HCl pH7.4, 0.125% Na deoxycholate, 0.375%Triton X-100, 0.15% NP-40, 4mM EDTA, 50 mM NaF) and immunoblotting was performed as previously described [3 (link)]. In brief, equal amount of proteins (15 ug) were resolved on 4-12% NuPAGE Bis-Tris gel and transferred to PVDF membrane. Pierce Reversible Protein Stain Kit was used to detect total proteins for loading normalization. Primary antibodies used in immunoblotting were phospho-S6(Ser235/236), S6, phospho-AKT (S473), phospho-AKT (T308), phospho-ULK (S757), ULK, phospho-AMPK (T172), AMPK, phospho-GSK-3β (S9), GSK-3β, Atg5 (all from Cell Signaling), phospho S657-PKCα, PKCα (both from Santa Cruz), LC3 (from Novus Biologicals), TFAM (mtTFA; from Abcam). Secondary antibodies used were donkey anti-rabbit IgG secondary antibody and goat anti-mouse IgG secondary antibody (both from Thermo Scientific. AlphaView Software was used for image acquisition and signal quantification.