Rabbit anti pcna

Western blotting was performed as described previously [8 (link)]. Briefly, normalized concentration protein was loaded on a 10% SDS–polyacrylamide gel and separated by electrophoresis. Then, the protein was transferred from the gel to a polyvinylidene difluoride (PVDF) membrane. After blocking with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), the membrane was probed using rabbit anti-Dicer (1:2000, Novus Biologicals, Littleton, CO, USA), rabbit anti-PCNA (1:1000, Cell signaling Technology), rabbit anti-phospho-Histone H3 (1:1000, Cell Signaling Technology), and mouse anti-β Actin (1:5000, Novus Biologicals, Lincoln, NE, USA) at 4 °C overnight. After washing with 1 × TBST, the membrane was incubated for 1 h at room temperature with IRDye 680 goat anti-rabbit IgG or IRDye 800 goat anti-mouse IgG (1:10,000, LI-COR Biosciences). The probed blot was scanned using an Odyssey infrared imager.

Cells were lysed in Tris-HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM, and protease inhibitors. Lysates were separated on 10% or 12% acrylamide gel and immunoblotted using standard procedures. Rabbit anti-OCT4, #2750 (Cell Signaling Technology Inc., Danvers, MA), rabbit anti-Nanog, PA1-097 (ThermoFisher Scientific, Rockford, IL), mouse anti-β-3-Tubulin (TU-20), #4466 (Cell Signaling Technology Inc.), mouse anti-GFAP, MAB360 (Merck Millipore, Darmstadt), rabbit anti-HspA1A, sc-33575 (Santa Cruz Biotechnology, CA), rabbit anti-PCNA, #13110 (Cell Signaling Technology Inc.), rabbit anti-NPM, #3542 (Cell Signaling Technology Inc.), mouse anti-GAPDH, ab8245 (AbCam, Cambridge, UK), rabbit anti-Hsp60, #D307 (Cell Signaling Technology Inc.), and HRP-conjugated secondary antisera (Santa Cruz Biotechnology, CA) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK) and images were acquired using BioRad ChemiDoc MP Imaging System (BioRad, Hercules, CA). Densitometric analysis was performed using the BioRad associated Image Lab Software (BioRad, Hercules, CA). Values are expressed as fold over internal control, represented by GAPDH or Hsp60, in the case of NPM, whose expressions were not significantly modulated in the proteome profiles.

The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Epo (ab65394) and rabbit anti-Ki-67 (ab15580) were purchased from Abcam (Cambridge, MA). Other chemicals and organic solvents of the highest available grade were obtained from Sigma-Aldrich. AMPKα1-KO and AMPKα2-KO mice were described elsewhere [55 (link), 56 (link)]. Mice were handled in accordance with study protocols approved by the Institutional Animal Care and Use Committee of University of Oklahoma Health Sciences Center (Oklahoma City, OK).

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Proteins were run on 8% or 12% polyacrylamide gel and transferred to a nitrocellulose membrane (#10600006, GE Helathcare Life science, DE). Membranes were blocked in TBS buffer (20 mM TRIS, 150 mM NaCl, pH 7.4) containing 5% Bovine Serum Albumin and exposed to rabbit-anti-mTOR (#2983, Cell Signalling, Danvers, MA, USA), rabbit-anti-phospho-mTOR (#5536, Cell Signalling), rabbit-anti-FN1 (#26836, Cell Signalling), rabbit-anti-phospho-Stat3 (#9145, Cell Signalling), rabbit-anti-Stat3 (#4904, Cell Signalling), rabbit-anti-Vimentin (#3932, Cell Signalling), rabbit-anti-GAPDH (#2118, Cell Signalling), rabbit-anti-PD-L1 (#13684, Cell Signalling), rabbit-anti-PCNA (#13110, Cell Signalling), rabbit-anti-N-Cadherin (#4061, Cell Signalling), and rabbit-anti-E-Cadherin (#3195, Cell Signalling) overnight at 4 °C. Membranes were washed in TBST (TBS-0.05% Tween-20) and incubated with either anti-rabbit HRP-conjugated secondary antibody for 1 h at room temperature. After several washes in TBST, the blots were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580, Thermo Fisher Scientific, Waltham, MA, USA).