Whole-cell extracts were prepared by lysing cells for 15 min on ice in RIPA lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0% NP-40, 0.1% SDS, and 0.1% Na-deoxycholic acid) supplemented with protease and phosphatase cocktail inhibitors (Complete and PhosSTOP; Roche Applied Science, Mannheim, Germany). Protein concentration in cell lysates was quantified by Bradford (BioRad) in 96 well plates using a Bio-Rad Benchmark Microplate Reader. Whole-cell lysates were separated through SDS-polyacrylamide gels (4–12%) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% milk powder in 0.1% Tween20 in PBS (PBS-T) for 1 hr at room temperature, followed by incubation with primary antibodies diluted in 2.5% milk PBS-T. ImmunoCruz Western Blotting Luminol Reagent (Thermoscience) was used to visualize protein levels with light sensitive-films (Phenix Research). The following antibodies were used: PD-1 (D4W2J, 1:250, Cell Signal Technology), and GAPDH and H2AX (1:1000, Santa Cruz Biotechnology)
Pd 1 d4w2j
Manufactured by Cell Signaling Technology
Sourced in United States
PD-1 (D4W2J) is a primary antibody that targets the programmed cell death protein 1 (PD-1). PD-1 is an immune checkpoint receptor expressed on the surface of T cells, B cells, and natural killer cells. The antibody can be used for the detection and study of PD-1 expression in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Complete and phosstop, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Benchmark microplate reader, by Bio-Rad (1 mentions)
Phospho mtor, by Abcam (1 mentions)
Phospho akt ser473, by Abcam (1 mentions)
S6 ribosomal protein, by Abcam (1 mentions)
β trcp, by Abcam (1 mentions)
Biosci new flash protein any kd page, by Dakewe (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
2 protocols using pd 1 d4w2j
1
Whole-Cell Lysis and Western Blot Analysis
2
Western Blot Analysis of Immune Checkpoint and Signaling Proteins
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To collect whole-cell lysates, cells were lysed in RIPA lysis buffer supplemented with a Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the cell lysates were centrifuged at 12,000 rpm. The supernatant was collected, mixed with loading buffer, and boiled for 15 min to disengage the protein secondary structure. The mixture was resolved on SDS-PAGE gels (BioSci™ NewFlash Protein AnyKD PAGE; DAKEWE, Beijing, China) and then transferred onto nitrocellulose membranes (GE life, Pittsburgh, PA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The bands were probed with the appropriate secondary antibodies and detected by enhanced chemiluminescence. The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): PD-1 (D4W2J , 1:1000), phospho-AKT (Ser473) (D9E, 1:2000), phospho-mTOR (Ser2448) (D9C2, 1:1000), phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, 1:2000), AKT (pan) (C67E7, 1:1000), mTOR (7C10, 1:1000), S6 Ribosomal Protein (54D2, 1:1000 ), β-TrCP (D13F10, 1:1000), and β-Actin (8H10D10, 1:1000 ), except anti-PD-L1 antibody [EPR19759] (ab213524; Abcam, UK, 1:1000).
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