Anti α tubulin

Frozen hearts, livers and kidneys were homogenized in modified RIPA buffer containing 50 mM Tris/HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxychoilate, 0.1% SDS, 1 mM EDTA, 10 mM sodium fluoride, Protease inhibitor cocktail (Roche Life Sciences, Mannheim, Germany) and Phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, Taufkirchen, Germany), pH 7.5 using an Ultra-Turrax T10 basic homogenizer. Samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes at 250 mA for 2 h and incubated with the following primary antibodies: anti-insulin receptor (1:1000, Cell Signaling Technologies, 3025), anti-NRF1 (1:2000, Abcam, ab175932). Anti-Rabbit IgG (H+L) Fab2 Alexa Fluor (1:5000; Cell Signaling, 4414) and anti-Mouse IgG (H+L) Fab2 Alexa Fluor (1:10000; Cell Signaling, 4408) served as secondary antibody. Detection and quantification of fluorescent bands was performed using the Bio-Rad Western Blot Imager. Loading control was performed using anti-alpha-tubulin (1:2000, Sigma-Aldrich, T9026). For blots investigating levels of MTHFD1L, ATP synthase β and alpha-tubulin performed using isolated mitochondria, the following primary antibodies were used: Anti-MTHFD1L (1:1000, Novus Biologicals NBP2-37864), anti-ATP synthase β (1:2000, BD Biosciences, 612519), anti-alpha-tubulin (1:2000, Sigma-Aldrich, T9026).

Chelidonine purchased from Sigma-Aldrich was used in this study. To inhibit apoptosis, the pan-caspase inhibitor Z-VAD-FMK (Enzo Life Sciences, Farmingdale, NY, USA) was used and dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used as a vehicle. For western blot analysis, the following antibodies were used: anticaspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP), Mcl-1, BAK, p-CDK1(Thr161), aurora A and p-PLK1 from Cell Signaling Technology (Beverly, MA, USA); anticyclin B1, CDK1, p-CDK1(Tyr15), and PLK1 from Santa Cruz Biotechnology (CA, USA); anti-BAX from BD Biosciences; anti-p-Ser/Thr-MPM-2 from Millipore; and anti-α-tubulin from Sigma-Aldrich. Anti-mouse IgG peroxidase conjugate and anti-rabbit IgG peroxidase conjugate from Sigma-Aldrich were used as secondary antibodies. For immunofluorescence assay, the following primary antibodies were used: anti-AIF from Santa Cruz, anti-α-tubulin from Sigma-Aldrich, and antipericentrin from abcam (Cambridge, MA, USA), and anti-mouse-FITC and anti-mouse-TRITC antibody were purchased from Sigma-Aldrich. To stain nucleus, we used DAPI from Sigma-Aldrich.