Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-RB (GeneTex, Irvine, CA, USA), anti-phospho-Akt(Ser473) (Cell Signaling), anti-Akt (Cell Signaling), anti-phospho-Erk1/2(Thr202/Tyr204) (Cell Signaling), anti-Erk1/2 (Cell Signaling), anti-TOP2A (Cell Signaling), anti-CCND1 (Cell Signaling), anti-γH2AX (Cell Signaling), anti-CDK2 (GeneTex), anti-RAD51 (GeneTex), anti-CDK4 (Cell Signaling), and anti-GAPDH (GeneTex) antibodies followed by horseradish peroxidase-conjugated secondary antibodies and visualization using an enhanced chemiluminescence detection system. Immunohistochemical (IHC) sample preparation and staining with anti-GFAP (Genetex), anti-phospho-RB (Cell Signaling), anti-Ki-67 (Cell Signaling), and anti-γH2AX (Cell Signaling) antibodies were carried out as previously described [48 (link)]. Uncropped Western blot images are provided in Figure S5 .
Anti phospho rb
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-Rb is a laboratory reagent that specifically detects the phosphorylated form of the Retinoblastoma (Rb) protein. Rb is a cell cycle regulatory protein whose phosphorylation status is indicative of cell cycle progression.
Automatically generated - may contain errors
Lab products found in correlation
Anti rb, by Cell Signaling Technology (7 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Anti cyclin d1, by Cell Signaling Technology (5 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti p21, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti cdk4, by Cell Signaling Technology (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Anti γh2ax, by Cell Signaling Technology (2 mentions)
Anti gapdh, by GeneTex (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti phospho cyclin d1, by Cell Signaling Technology (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti p21, by Santa Cruz Biotechnology (2 mentions)
Anti phospho cdk2, by Cell Signaling Technology (2 mentions)
Anti phospho p53, by Cell Signaling Technology (2 mentions)
Anti cc3, by Cell Signaling Technology (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti bcl xl, by Abcam (2 mentions)
22 protocols using anti phospho rb
1
Immunoblotting and Immunohistochemical Analysis
2
Investigating Obinutuzumab and Doxorubicin Effects
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Cells were seeded on 6-well plates at 1 Â 10 6 cells/well or 25 cm 2 flask at 3 Â 10 6 cells/flask, and treated with obinutuzumab and/or doxorubicin or prednisolone at indicated concentrations. Cells were lysed with cell lysis buffer (Cell Signaling Technology) containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Nacalai Tesque, Inc.). Cell lysates were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes by using an iBlot Gel Transfer System (Invitrogen). Immunoblotting was performed using the following primary antibodies: anti-IRE1, anti-phospho-JNK (pJNK), anti-phospho-Bcl2 (pBcl2), anti-Bcl2, anti-phospho-Rb at Ser807/Ser811 [pRb (Ser807/811)], anti-phospho-Rb at Ser795 [pRb (Ser795)], antiphospho-Rb at Ser780 [pRb (Ser780)], anti-p27, anti-Skp2, and anti-b-actin (Cell Signaling Technology), and anti-phospho-IRE1 (pIRE1), anti-JNK, and anti-Rb (Abcam). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), followed by chemiluminescence detection.
3
Immunoblotting of Autophagy and Apoptosis Markers
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Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-p62 (GeneTex, Irvine, CA, USA), anti-LC3B (Novus biologicals, Littleton, CO, USA), anti-caspase3 (Novus biologicals), anti-γH2AX (Cell Signaling), anti-PIK3C3 (GeneTex) and anti-GAPDH (GeneTex) antibodies, followed by visualization using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence detection system.
4
Molecular Mechanisms of Glioblastoma Response to Temozolomide
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Reagent and antibody sources were as follows: AG1478 (Calbiochem/Merck, Darmstadt, Germany), BMP4 (R&D Systems, Minneapolis, MN, USA), DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), temozolomide (TMZ) and anti-β-Actin-peroxidase conjugated antibody (Sigma-Aldrich, Munich, Germany), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-BIM, anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cleaved PARP (poly (ADP-ribose) polymerase-1), anti-EGF Receptor, anti-phospho-EGF receptor (Tyr1068), anti-FOXO3a, anti-phospho-FOXO3a (Thr32), anti-phospho-FOXO3a (Ser253), anti-phospho-FOXO3a (Ser318/321), anti-phospho-Rb (Ser807/811), anti-SMAD1, anti-SMAD3, anti-SMAD4, anti-SMAD5, anti-phospho-SMAD1/5 (Ser463/465), anti-phospho-SMAD3 (Ser423/425), anti-p27Kip1, anti-SOX2 (Cell Signaling Technology, Beverly MA, USA), anti-OLIG2, anti-β-Tubulin beta III isoform (Millipore, Temecula, CA, USA), anti-CYCLIN B1, p21CIP1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-GFAP (BD Pharmingen San Jose, CA), anti-NESTIN (R&D Systems, Minneapolis, MN, USA), and anti-CYCLIN D1 (ThermoFisher Scientific, Waltham, MA USA).
5
Western Blot Analysis of Cell Cycle Regulators
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Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked with fat‐free milk combined with tris‐buffered saline plus tween 20 for one hour at room temperature and then incubated with the appropriate primary antibody and horseradish peroxidase conjugated secondary antibodies. Imager was used to visualize the blots. The primary antibodies used in this study were as follows: anti‐CDK4 (#12790, 1:1000, cell signaling), Anti‐Cyclin D1 antibody (#2978, 1:1000, cell signaling), Anti‐phospho‐EGFR antibody (#47724, 1:1000, cell signaling), Anti‐Phospho‐RB (ser780, #9307, 1:1000, cell signaling), anti‐E2F1 (#666515,1:1000,Proteintech), CDKN2a (#80772, 1:1000, cell signaling); and β‐actin (ab92552, 1:1000).
6
MEDICA Synthesis and Signaling Pathway Analysis
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MEDICA [α,α′-tetramethyl hexadecanedioic acid, HOOC-C(CH3)2 -(CH2)12-C(CH3)2-COOH] was synthesized as previously described [21 (link)]. Anti-p21/WAF1 (#2946), anti-Cyclin D1(#2922), anti-phospho-RB(#9308), anti-β-Actin (#4967), anti-PARP (#9542), anti-cleaved Caspase3 (#9661), anti-phospho-Akt(Ser473) (#4060), anti-Akt (#9272), anti-phospho-STAT3(Tyr705) (#9145), anti-phospho-EGFR(Thr669) (#3056), anti-phospho-LKB1(Ser428) (#3051), anti-LKB1(#3050), anti-phospho-CREB(Ser133) (#9191), anti-CREB (#9197), anti-phospho-IRS-1(Ser636/639) (#2388), anti-phospho-ACC(Ser79)(#3661) antibodies were from Cell Signaling. Anti-EGFR(sc-03), anti-phospho-Erk(Tyr204) (sc-7383), anti-Erk (sc-93), anti-Met (sc-10), anti-STAT3 (sc-8019), anti-phospho-RSK1/2(Thr359/Ser363) (sc-12898), anti-RSK(sc-231), anti-phospho-STAT3(Ser727) (sc-8001), anti-IRS-1(sc-559) antibodies were from Santa Cruz Biotechnology. Anti-phospho-EGFR(Tyr1068) (ab40815), anti-phospho-HER(Tyr1248) (ab47755) antibodies were from Abcam. Anti-phospho-FAK(Ser910) (#445596) antibody was from Invitrogen. Anti-FAK(#61087) antibody was from BD. Anti-gp130 (#29731) antibody was from Upstate Biotechnology. Anti-α-Tubulin (T6074) antibody was from Sigma. EGF and HGF were from PeproTech. IL6 and IL6R were from R & D Systems. U0126 and PD0325901 were from Sigma.
7
Profiling Kinase Signaling Pathways
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Cultured cells in log-phase growth were treated with 100 nM of MEK162 for 10 min, 24 and 48 h. The plates were then washed twice with ice-cold PBS, lysed and collected using mild lysis buffer. Lysates were centrifuged at 10 000 r.p.m. at 4 °C for 10 min to clear insoluble material, and the resulting supernatant was collected and quantified using a bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL, USA). Protein was resolved by SDS–PAGE and transferred to nitrocellulose membranes (Invitrogen). Anti-phospho-AKT (Thr308 cat # 9275), anti-total AKT (cat # 92725), anti-phospho-ERK1/2 (T202/Y204 cat # 9101), anti-total ERK1/2 (cat # 9102), anti-phospho-S6 ribosomal protein (Ser235/236 cat # 2211), anti-total S6 Ribosomal Protein (SG10 cat # 2217), anti-phospho-RB (Ser780 cat # 9307), anti-total RB (cat # 9309), anti-cyclin D1(cat # 2921), anti-phospho cyclin D1 (cat # 2922), anti-phospho-FoxO1(Thr24)/FoxO3a (Thr32) (cat # 9464), anti-p27kip (cat # 2552), anti-beta-actin (cat # 4967), and anti-alpha-tubulin (cat # 2144) antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA).
8
Immunoblotting of Apoptosis and Cell Cycle Proteins
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Cells were lysed in lysis buffer [50 mmol/L Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP40] in the presence of protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails I and II (Sigma). Equal amounts of protein, as determined by the Bradford assay, were resolved by electrophoresis through an SDS 10% or 12.5% polyacrylamide gel and then transferred to a PVDF membrane (Millipore). The membrane was incubated with one of the following primary antibodies: anti-cleaved CASPASE-3 (#9664S, Cell Signaling); anti-TUBULIN (T9026, Sigma); anti-GAPDH (sc-25778, Santa Cruz Biotechnology); anti-phospho-RB (#9308, Cell signaling); [5 (link)]-anti-p21 (sc-397, Santa Cruz Biotechnology). Binding of the primary antibody was detected using an enhanced chemilluminescence kit (ECL Amersham).
9
Immunofluorescence Staining of Cell Cycle Markers
10
Western Blot Protein Analysis Protocol
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Cells were directly lysed in Laemmli buffer (25 mM Tris-HCl pH 6.8; 2% SDS; 10% glycerol; 2.5% β-mercaptoethanol; 0.01% bromophenol blue). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes (88018, Thermo Fisher Scientific). Membranes were blocked with 5% non-fat dried milk or 5% BSA in PBS. Then, membranes were incubated overnight with the primary antibody at the dilution recommended by the manufacturer: anti-PAR (AM80, Calbiochem), anti-GAPDH (sc-32233, Santa Cruz Biotechnology), anti-Rb (#9309, Cell Signaling), anti-phospho-Rb (#9308, Cell Signaling), and anti-p16 (550834, BD Pharmingen). Secondary antibodies used were anti-mouse and anti-rabbit peroxidase conjugated (respectively 715-035-151, 711-035-152, Jackson ImmunoResearch Laboratories) or anti-mouse fluorescent dye conjugated secondary antibody (926-32210, LI-COR). The peroxidase activity was revealed using an ECL kit (RPN2106, Amersham Biosciences) or SuperSignal West Dura Extended Duration Substrate (34076, Thermo Fisher Scientific). For fluorescent Western blots, the total proteins used for normalization were detected by using the Revert 700 nm Total Protein Stain (926-110.21, LI-COR).
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