Rabbit anti hif 1α

Total protein was extracted from tissues by RIPA, and the concentration was measured with BCA Protein Quantitative kit. After denaturing by boiling for 5–10 min, the proteins were loaded, separated by SDS-PAGE gel electrophoresis and transferred onto nitrocellulose filter (NC) membranes. After blocked with 5% skim milk or 5% Bovine albumin (BSA), the NC membranes were incubated with the following antibodies at 4°C overnight: rabbit anti-SIK2 (Thermo Fisher; 1:1000); rabbit anti-HIF-1α (Bioss; 1:1000); rabbit anti-mTOR (Cell Signaling; 1:1000); rabbit anti-p-mTOR (Cell Signaling; 1:1000); rabbit anti-PINK1(Abcam; 1:1000); mouse anti-Parkin (Abcam; 1:1000). After rinsed with Tris-buffered saline with Tween (TBST), these membranes were incubated with goat anti-rabbit or anti-mouse IgG-HRP (Biosharp; 1:10000), and exposed with ECL reagent (Biosharp). β-actin was used as the endogenous control.

The immunofluorescence studies were performed as described previously [18 (link)]. The cells were fixed, permeabilized, and incubated overnight at 4 °C with the following respective primary antibodies: rabbit anti-Fbp2 (1:500, produced and tested as described previously [19 (link)]), mouse anti-Fbp2 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-271799), rabbit anti-Hif1α (1:500, Bioss Antibodies, Woburn, MA, USA, bs-0737R), rabbit anti-Ldha (1:500, Novus Biologials, Littleton, CO, USA, NBP1-48336), rabbit anti-Hk2 (1:500, Merck ab3279), rabbit anti-Ki67 (1:500, Abcam, Cambridge, UK, ab15580), mouse anti-αSMA (1:500, Merck a5228), mouse anti-β-actin (1:500, Sigma-Aldrich a1978), and rabbit anti-β-actin (1:500, Sigma–Aldrich a2066). The primary antibodies were detected using the following fluorophore-labelled secondary antibodies: goat anti-mouse-AlexaFluor488 (1:1000, Abcam, ab150113), goat anti-mouse-AlexaFluor633 (1:1000, Thermo Fisher Scientific, Waltham, MA, USA, a21050), goat anti-rabbit-AlexaFluor 488 (1:1000, Invitrogen a11034), and goat anti-rabbit-AlexaFluor633 (1:1000, Invitrogen a21070). Nuclei were counterstained with DAPI. In the controls, the primary antibodies were omitted.