Anti tsc1

Proteins were extracted from RMCs using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, and protease cocktail at 1 μg/mL). Protein concentrations were determined using a BCA kit (Thermo Scientific, Rockford, AL, USA). Equal amounts of protein (60 µg) were separated by SDS-PAGE on a 6%, 10%, or 12% acrylamide gel and then transferred onto a nitrocellulose membrane. After the membrane had been blocked with non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature, it was probed with the following primary antibodies overnight at 4°C: anti-VDR (catalogue no.: ab109234, Abcam, USA), anti-DDIT4 (catalogue no.: NBP1–77321, Novus Biologicals, USA), anti-TSC1 (catalogue no.: 6935S, Cell Signaling Technology, USA), anti-TSC2 (catalogue no.: 4308P, Cell Signaling Technology), anti-Rheb (catalogue no.: 13879S, Cell Signaling Technology), anti-mTOR (catalogue no.: Ab51044, Abcam), anti-4E-BP1 (catalogue no.: ab2606, Abcam), and anti-p70S6K (catalogue no.: ab32359, Abcam). After extensive washing, the membranes were incubated with Dylight anti-rabbit IgG secondary antibody. The antigens were visualized using the Odyssey infrared imaging system (LI-COR Biotechnology, Nebraska, USA). The results were expressed as the relative intensity (RI) intensity (adjusted to that of β-actin) of each band.

CDS encoding PIWIL2 and p65 was synthesized and inserted into pcDNA3.1 and pEGFP, respectively. shRNA against PIWIL2 and p65 were synthesized by TSINGKE Biological Technology company (Beijing, China) and the target sequences of these shRNA were as follows:
Human PIWIL2 shRNA (shPIWIL2-1): 5′-CGG ATT GAG GAG AAA CGT AAA CTC-3′
Human PIWIL2 shRNA (shPIWIL2-2): 5′-CTA TGA GAT TCC TCA ACT ACA GAA G-3′
Human p65 shRNA (shp65): 5′ -CGG ATT GAG GAG AAA CGT AAA CTC-3′
Anti-PIWIL2, anti-p62, anti-Beclin-1, and anti-LaminB1 were purchased from Santa Cruz (USA). Anti-IKKα, anti-IKKβ, anti-p-IKKα/β(Ser176/180), anti-IκBα, anti-p-IκBα(Ser32/36), anti-p65, anti-p-mTOR(Ser2448), anti-p-ULK1(Ser555), anti-p-4E-BP1(Thr37/46), anti-p-P70S6(Thr389), and anti-TSC1 were purchased from Cell signaling technology (USA). Anti-LC3B was purchased from Novus Biologicals (USA). Anti-mTOR and anti-Bcl-2 were purchased from Proteintech Group (USA). Anti-GAPDH was purchased from Epitomics (USA).