Cells were lyzed with RIPA buffer supplemented with protease and phosphatase inhibitors (A32959, Thermo). Cell extracts were separated in SDS‐PAGE and then transferred to PVDF membranes. The primary antibodies were incubated overnight at 4°C, and the HRP‐conjugated secondary antibodies were incubated for 1 h at room temperature. The chemiluminescent signals were detected with the ECL Plus Detection Reagents (WBULS0500, Millipore). Antibodies against Spt16 (8D2) was purchased from Biolegend. Antibodies against p‐ATR (2853), p‐ATM (5883), p‐BRCA1 (9009), p‐CHK2 (Thr1079, 8654), N‐cadherin (13116), E‐cadherin (3195), ZO‐1 (13663), vimentin (5741), Snail (3879), Bcl‐2 (4223), cleaved caspase 7 (8438), and cyclin D1 (55506) were purchased from Cell Signaling Technology. Antibodies against p‐Rb (sc‐377539), cyclin E (sc‐377100), MCM7 (sc‐9966), and Rb (sc‐102) were purchased from Santa Cruz Biotechnology. Antibodies against GAPDH (HRP‐6004) and β‐actin (HRP‐60008) were purchased from Proteintech.
Rb sc 102
Manufactured by Santa Cruz Biotechnology
Rb (sc-102) is a rabbit polyclonal antibody produced by Santa Cruz Biotechnology. It is designed for use in various immunological techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
A32959, by Thermo Fisher Scientific (1 mentions)
β actin hrp 60008, by Proteintech (1 mentions)
Cyclin d1 55506, by Cell Signaling Technology (1 mentions)
Mcm7 sc 9966, by Santa Cruz Biotechnology (1 mentions)
P rb ser780, by Cell Signaling Technology (1 mentions)
3 protocols using rb sc 102
1
Protein Expression and Signaling Analysis
2
Immunohistochemical Analysis of Lung Cancer
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This work was approved by the Ethics Review Committee of the First Affiliated Hospital of Xi’an Jiaotong University. A total of 147 lung cancer specimens and adjacent normal tissues were obtained from the First Affiliated Hospital of Xi’an Jiaotong University. After antigen retrieval in sodium citrate buffer (PH = 9.0) in a microwave oven, the paraffin‐embedded tissues were stained with Spt16 (8D2, Biolegend), MCM7 (sc‐9966, Santa Cruz), and Rb (sc‐102, Santa Cruz) antibodies following the manufacturer’s protocol for the LSAB detection kit (ZSGB‐BIO). Each section was scored by the staining intensity (0, none; 1, weak; 2, moderate; and 3, strong) and the positive cell rate (0, no positive cells; 1, 10%–25%; 2, 25%–50%; 3, 50%–75%; 4, >75%). The total score is the product of the positive cell rate by staining intensity.
3
Protein Expression Analysis in Cell Lines
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Proteins from cells treated for 72 h with corresponding IC50 concentration for respective cell lines and control cells were extracted using RIPA lysis buffer supplemented with proteinase and phosphatase inhibitors. Protein amount was quantified using the Pierce BCA Protein assay kit (Thermo Fisher Scientific). The same amount of total protein sample (20 µg) was separated from each sample using SDS-PAGE and transferred onto 0.2-µm PVDF membrane. In order to optimize the number of proteins analyzed from a single membrane the blots were cut in multiple pieces according to size. Uncropped blot images are presented in Supplementary Fig. 9 . The blots were probed overnight at 4 °C with the following antibodies: CDK4 (sc-56277), CDK6 (sc-7961), MDM2 (sc-965), p53 (sc-126), PUMA (sc-374223), and RB (sc-102) from Santa Cruz Biotechnology (Dallas, TX); p-RBSer807/811 (#702097) from Thermo Fisher Scientific; and p-RBSer780 (#9307), p21 (#2947), and KU80 (#2573) from Cell Signaling Technology (Danvers, MA). Protein levels were quantified using ImageJ software60 (link) with normalization against KU80.
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