P prb

Primary and immortalized HDPCs were seeded in 6-well plates and incubated until they reached 100%confluence. Harvested cells were washed with phosphate-buffered saline (PBS) and lysed in buffer containing 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 1 mM ethylene diamine tetra-acetic acid, 1% NP40, 0.4 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate, 1 mM ethylene glycol tetra-acetic acid, 50 mM Tris (pH 7.4), and aprotinin (10 μg/ml) at 4°C for 30 min. Equal amounts of the quantified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. Specific primary antibodies against p53 (#NB200-171) (Novus Biologicals, Centennial, USA), cyclin E (#sc-481) (Santa Cruz Biotechnology, USA), p21(#2947T), CDK2 (#2546T), pRb (#9309s), and p-pRb (#8147s) (Cell Signaling Technology, USA) were used. Densitometric graphs of three independent experiments were generated using ImageJ software (version 1.5) [15 (link)]. Band intensities were normalized to those of β-actin or GAPDH.

17-Hydroxyprogesterone (OHPg), aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMFS), sodium orthovanadate, NaCl, MgCl2, EGTA, glycerol, Triton X-100, Fetal Calf Serum (FCS), Fetal bovine serum (FBS), HEPES were from Sigma-Aldrich (Milan-Italy). Antibodies against human PR, β-actin, p27, Bcl-2, p21, p53, RNA Pol II, SRC-2, p-JNK, p-MAPK (ERK1, ERK2), JNK, MAPKs (ERK1, ERK2) and Protein A/G PLUS-Agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to p-p38, p-38, p62, p-pRb and Beclin-1 were from Cell Signaling (Beverly, MA). RU 486, SB203580, PD98059, SP600125 were from Calbiochem.

CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay Reagent [MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was purchased from Promega (Madison, WI, USA). Propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) stain were purchased from Sigma-Aldrich (St. Louis, MO, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents were purchased from Pierce (Rockford, IL, USA). Antibodies specific to PARP, caspase-3, caspase-8, p53, Bcl-2, Bcl-xL, Bax, Bid, pRb, p-pRb, and cytochrome C were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and anti-mouse IgG HRP-conjugated secondary antibody were purchased from Millipore (Billerica, MA, USA). Antibodies specific to p27, p21, and glyceraldehyde 3-phospahte dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolycarbocyanine chloride) was purchased from Enzo (Farmingdale, NY, USA). General-caspase inhibitor Z-VAD-fmk and caspase-8 inhibitor Z-IETD-fmk were purchased from R&D systems (Minneapolis, MN, USA). The FITC-Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Jose, CA, USA).

Human GBM cell lines U251 and U87 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences for this study. These cell lines were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified incubator with 5% CO₂. Antibodies against MALT1, p‐pRb, cyclin D1, CDK4, p27, p21, p65, IκB‐α and lamin A/C were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). Antibodies specific to β‐actin were purchased from Abcam (Cambridge, MA, USA). DAPI was obtained from Sigma‐Aldrich Chemical Co. (St. Louis, MO, USA).

PC12, HT-29, HEK293, and 293T cells were obtained from the National Infrastructure of Cell Line Resources, China. The hPheo1-Tet-GIPC2 or PC12-Tet-GIPC2 cells were stable cell lines custom made by Genecopoeia, China, and tetracycline or Doxycycline (3 μg/mL) was added to induce overexpression of GIPC2. Primary rat adrenal chromaffin cells were isolated from the adrenal glands according to the protocol of Domínguez et al.^{41 (link)}, with the modification that only 5- to 7-days rats were used. All cells were cultured according to the guideline of American type culture collection (ATCC).
The primary antibodies against pERK1/2, ERK1/2, pMEK, MEK, p27, ACTB, p-pRB, p53, HA-Tag were purchased from Cell Signaling Technology (USA), antibodies against NONO, HIF1A, HIF2A, VHL were purchased from Abcam (UK), antibodies against p18, pRB were purchased from Santa Cruz (USA), GIPC2, and RET antibodies were purchased from LSBio (USA) and Abbiotec (USA) respectively. GIPC2 siRNA were purchased from Dharmacon. SDHD and VHL siRNAs^{11 (link)} were synthesized by Invitrogen. For the RNAi experiment, siTran1.0 Reagent (Origene, USA) was used according to the manufacturer’s instructions.

FAK (catalog no.: 05-537; mouse), PCNA (catalog no.: PLA0080; rabbit), GST (catalog no.: 05-782), ubiquitin (catalog no.: ST1200), and GAPDH (catalog no.: MAB374; mouse) antibodies were purchased from Millipore; pY397 FAK (catalog no.: 44-624G; rabbit) and cyclin D1 (catalog no.: MA5-16356; rabbit) antibodies were purchased from Life Technologies; CDK4 (catalog no.: sc-166373; mouse), CDK6 (catalog no.: sc-7961; mouse), CDK1 (catalog no.: sc-54; mouse), CDK2 (catalog no.: sc-6248), CDH1 (catalog no.: sc-56312; mouse), Myc (catalog no.: sc-40; mouse), and cyclin D3 (catalog no.: sc-6283; mouse) antibodies were purchased from Santa Cruz Biotechnology; p-pRb (catalog no.: 8516; rabbit) antibody was purchased from Cell Signal Technology; cyclin D2 (catalog no.: 554201; mouse) antibody was purchased from BD Biosciences; GFP (catalog no.: MMS-118P) antibody was purchased from Covance; FAK-I PF-271 was purchased from MedKoo; MG-132 was purchased from Selleckchem; cycloheximide and FLAG (catalog no.: F3165; mouse) antibody were purchased from Sigma; and leptomycin B was purchased from LC Laboratories.