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Pbads112

Manufactured by Cell Signaling Technology
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PBADS112 is a lab equipment product from Cell Signaling Technology. It is a device used for sample preparation and analysis in biological research.

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Lab products found in correlation

Cleaved parp, by Cell Signaling Technology (3 mentions) P 4e bp1 t37 46, by Cell Signaling Technology (2 mentions) 4e bp1, by Cell Signaling Technology (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Protease inhibitor, by Roche (1 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions) Protein assay dye reagent concentrate, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Hif 2α, by Cell Signaling Technology (1 mentions) P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions) Pakt t308, by Cell Signaling Technology (1 mentions) Ps6 s235 236, by Cell Signaling Technology (1 mentions) P p90rsks380, by Cell Signaling Technology (1 mentions) Rsk1 2 3, by Cell Signaling Technology (1 mentions) Pbads136, by Cell Signaling Technology (1 mentions) Actin hrp, by Santa Cruz Biotechnology (1 mentions) Pegfr y1068, by Cell Signaling Technology (1 mentions) Hif 1α, by Cell Signaling Technology (1 mentions) Celltiter glo luminescent assay, by Promega (1 mentions)

5 protocols using pbads112

1

Western Blot Analysis of Signaling Proteins

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Cells were lysed in RIPA buffer complemented with Set I and Set II phosphatase inhibitors at 1× (Calbiochem), and protease inhibitors at 1× (Roche). Whole cell lysate concentration was determined with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Proteins were resolved on SDS-PAGE gels and electrotransferred to nitrocellulose membranes, 0.2 µm (Bio-Rad). Primary antibodies pS6S235/236, S6, pAKTS473, AKT, p4E-BP1T37/46, 4E-BP1, Cleaved PARP, pAktT308, p62, Rictor, Raptor, HIF-1α, HIF-2α, pERK1/2T202/Y204 (mouse), ERK, p-p90RSKS380, RSK1/2/3, pBADS112, pBADS136, pEGFRY1068, cleaved-caspase3 were from Cell Signaling Technologies. VHL (Santa Cruz #FL-181). mTOR primary antibody was from Millipore. Primary antibody dilutions were to manufactures' specifications (See Table S1). Tubulin (Sigma #T5168), KU-80 (GeneTex #GTX70485) and Actin-HRP (Santa Cruz #C-11) primary antibodies served as loading controls (LC) where noted. Secondary anti-Rabbit and ant-mouse antibodies were from (Fisher) and diluted in 5% milk, 1× TBS-T solution. ECL Western Blotting Detection reagents (GE Healthcare) were used for developing blots onto autoradiography film. For difficult to detect proteins SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used in combination with ECL.
Bailey S.T., Zhou B., Damrauer J.S., Krishnan B., Wilson H.L., Smith A.M., Li M., Yeh J.J, & Kim W.Y. (2014). mTOR Inhibition Induces Compensatory, Therapeutically Targetable MEK Activation in Renal Cell Carcinoma. PLoS ONE, 9(9), e104413.
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2

Potent Anticancer Effects of FRAX1036

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FRAX1036 was synthesized by Afraxis, Inc. (La Jolla, CA, USA) and docetaxel was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies used for immunoblotting (p-MEK1-S298, p-CRAF-S338, Cleaved PARP, Cyclin D1, p-Stathmin-S16, p-β-catenin-S675, MCL-1, BCL-xL, p-Bad-S112 and PAK1) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-Actin was purchased from Sigma (St Louis, MO, USA). Cell lines were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained at 37°C and 5% CO2 in RPMI 1640 media with 10% fetal bovine serum and 2 mM L-glutamine. U2OS-red fluorescent protein (RFP)-Tubulin cells (Marinpharm, Luckenwalde, Germany) were stably transduced with a plasmid expressing green fluorescent protein (GFP)-histone H2B. Cell transfections and treatments were performed using short interfering RNA oligonucleotides for PAK1 from Dharmacon RNAi Technologies (Chicago, IL, USA). Cellular viability was assessed via ATP content using the CellTiter-Glo Luminescent Assay (Promega, Madison, WI, USA) and results represent mean ± standard deviation from three experiments.
Ong C.C., Gierke S., Pitt C., Sagolla M., Cheng C.K., Zhou W., Jubb A.M., Strickland L., Schmidt M., Duron S.G., Campbell D.A., Zheng W., Dehdashti S., Shen M., Yang N., Behnke M.L., Huang W., McKew J.C., Chernoff J., Forrest W.F., Haverty P.M., Chin S.F., Rakha E.A., Green A.R., Ellis I.O., Caldas C., O’Brien T., Friedman L.S., Koeppen H., Rudolph J, & Hoeflich K.P. (2015). Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents. Breast Cancer Research : BCR, 17(1), 59.
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3

Western Blot Analysis of Cellular Signaling

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Cells were treated as indicated and western blot analysis was performed as previously described [2 (link)]. The pS6 S235/236 (4858), pS6 S240/244 (2215), S6 (2317), 4EBP1 (9644), pRb S780 (8180), pRb S807/811 (8516), Rb (9309), pBAD S112 (5284), p-p70S6K T389 (9205), p70S6K (2708), PIM1 (3247), PIM2 (4730), PIM3 (4165), TSC2 (4308), pGSK3β S9 (5558), GSK3β (9315), pPRAS40 T246 (2997), PRAS40 (2691), and GAPDH (2118) primary antibodies were purchased from Cell Signaling Technology, while the CDK4 (ab75511) and CDK6 (ab124821) antibodies were from Abcam and β-actin (A5441) was from Sigma-Aldrich. IRDye secondary antibodies were from LI-COR.
Litchfield L.M., Boehnke K., Brahmachary M., Mur C., Bi C., Stephens J.R., Sauder J.M., Gutiérrez S.M., McNulty A.M., Ye X.S., Wu W., Lallena M.J., Gong X., Merzoug F.F., Jansen V.M, & Buchanan S.G. (2020). Combined inhibition of PIM and CDK4/6 suppresses both mTOR signaling and Rb phosphorylation and potentiates PI3K inhibition in cancer cells. Oncotarget, 11(17), 1478-1492.
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4

Western Blot Analysis of Cellular Signaling

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Compounds were supplied by in-house synthesis at Genentech, Inc. or purchased from vendors. Antibodies used from immunoblots to AKT (#2920, 1:1000), AKT1 (#2938, 1:1000), AKT2 (#3063, 1:1000), AKT3 (#8018, 1:1000), pAKT(T308) (#2965, 1:1000), pAKT(S473) (#9271, 1:1000), pPRAS40(T246) (#2997, 1:1000), PRAS40 (#2691, 1:1000), pS6(S235/236) (#2211, 1:1000), S6 (#2317, 1:1000), p4EBP1 (T37/46) (#2855, 1:1000), p4EBP1 (S65) (#9456 1:1000), 4EBP1 (#9452, 1:1000), PARP (#9532 1:1000), Cleaved PARP (#5625, 1:1000), PTEN (#9556, 1:1000 and #9559, 1:1000), PIM2 (#4730, 1:500), PIM3 (#4165, 1:500), pGSK-3β (S9) (#9336, 1:500), GSK-3β (#9832, 1:500), pBAD (S112) (#9239, 1:500), and BAD (#9239, 1:500) were obtained from Cell Signaling Technology. The PIM1 antibody was obtained from Abnova (H00005292-M01, 1:500). An additional antibody to total PRAS40 was obtained from Invitrogen/ThermoFisher (AHO1031, 1:1000). Protein loading was assessed using antibodies to β-actin (Sigma-Aldrich, A5441, 1:3000), β-Tubulin (Sigma-Aldrich, T8328, 1:5000) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Advanced ImmunoChemical, 2-RGM2, 1:2000).
Savill K.M., Lee B.B., Oeh J., Lin J., Lin E., Chung W.J., Young A., Chen W., Miś M., Mesh K., Eastham J., Gnad F., Jiang Z., Stawiski E.W., Haley B., Daemen A., Wang X., Koeppen H., Modrusan Z., Martin S.E., Sampath D, & Lin K. (2022). Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors. Nature Communications, 13, 2057.
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5

Combination Therapy Evaluation Protocol

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SGI-1776, ruxolitinib, and bortezomib were obtained from Selleck Chemicals. AZD1208 was obtained from AstraZeneca, Inc. All drugs were solubilized in DMSO and stored at −20 or −80°C. Antibodies used in this study were: phospho (P)-p70S6K (T389) (#9234S); P-BAD (S112) (#9296); P-4EBP1 (T37/46) (#2855); P-S6 (S235/236) (#4858); BAD (#9329); hu-cl-PARP (#5625); mm-cl-PARP (#9544); GAPDH (#5174) (Cell Signaling Technology); Tubulin (#SC-5286); (Santa Cruz Biotechnology); and actin (#A5316) (Sigma-Aldrich). Antibody for mouse MDM2 was a gift from Jiandong Chen (Moffitt Cancer Center) and was previously described [75 (link)]. For immunoblotting, protein concentration was determined using Pierce™ BCA Protein Assay kit (Thermo Scientific) and a Benchmark Plus Microplate Spectrophotometer (Bio-Rad), and immunoblots were performed by standard SDS-PAGE. Horseradish peroxidase-conjugated secondary antibodies were from Thermo Scientific. Blots were developed using chemillumination detection reagents (Thermo Scientific).
Mazzacurati L., Lambert Q.T., Pradhan A., Griner L.N., Huszar D, & Reuther G.W. (2015). The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells. Oncotarget, 6(37), 40141-40157.
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