Anti ndrg1

Preparation of cell lysates and immunoblot analysis was performed via established protocols [82 (link)]. Anti-human primary antibodies were implemented at a 1:1, 000–2, 000 dilution and include: anti-NDRG1 (Cat#:ab37897) from Abcam (Cambridge); anti-Src (Cat#:2123), anti-phospho-Src Family (Tyr416; Cat.#:6943), anti-phospho-Src(Tyr527; Cat.#:2105), anti-p130Cas (Cat.#:13846), anti-phospho-p130Cas (Tyr249; Cat.#:4014), anti-phospho-p130Cas (Tyr410; Cat.#:4011), anti-EGF Receptor (Cat.#:2926), anti-phospho-EGF Receptor (Tyr1148; Cat.#:4404), anti-c-Abl (Cat.#:2862), anti-phospho-c-Abl (Tyr245; Cat.#:2861), anti-PAK1 (Cat.#:2602), anti-phospho-PAK1 (Thr423; Cat.#:2606), anti-PTP-PEST (Cat.#:4864), anti-PTP1B (Cat.#:5311), anti-CrkII (Cat.#:3492) and anti-phospho-CrkII (Tyr221; Cat.#:3491) were from Cell Signaling Technology (Boston, MA); and anti-Rac1 (Cat.#:05–389) was from Millipore (Darmstadt). The secondary antibodies (1:10, 000 dilution) included: anti-goat (Cat.#:A5420), anti-rabbit (Cat.#:A6154) and anti-mouse (Cat.#:A4416) antibodies from Sigma-Aldrich. β-actin (1:10, 000; Cat.#:A1978, Sigma-Aldrich) was used as a loading control.

EDHB was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in ethanol for biochemical assays. Anti-AKR1C antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-3 and anti-caspase-9 antibodies were procured from Enzo Life Sciences (NY). Anti-LC3A/B and anti-Beclin antibodies were purchased from Cell Signaling Technology (Boston, MA). Anti-β-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Anti-BNIP3 and anti-NDRG1 antibodies were purchased from Abcam (Abcam, MA).

Cells were collected and placed on ice, and lysed by RIPA lysis buffer (Beyotime, China) containing protease inhibitors and phosphatase inhibitors. Equal amounts of cell lysate (20 μg of protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, United States). Western blot analysis was performed with the following primary antibodies: anti-β-tubulin, anti-p44/42 MAPK (Erk1/2) and anti-MDR1/ABCB1 (1:1000 dilution; Cell Signaling Technology, United States), anti-BMP6, anti-TLE3 and anti-NDRG1 (1:5000 dilution; Abcam, United Kingdom), anti-Phospho-ERK1/2 (Thr202/Tyr204) (1:1000 dilution; Genxspan, China).