Western Blot Analysis of CD44 in Tumor Samples

Cell lysate was generated from tumor samples as described in the Registered Report (Li et al., 2015 (link)). Membranes were loaded with 40 µg of total protein and probed with: rabbit anti-CD44 (AbCam, cat# ab51037, clone EPR1013Y, RRID:AB_868936), 1:1000 dilution; mouse anti-ß-actin (Cell Signaling Technology, cat# 3700, clone 8H10D10, RRID:AB_2242334), 1:1000 dilution and the appropriate secondary antibody: HRP-conjugated goat anti-rabbit (Cell Signaling Technology, cat# 7074, RRID:AB_2099233), 1:2000 dilution; HRP-conjugated horse anti-mouse (Cell Signaling Technology, cat# 7076, RRID:AB_330924), 1:5000 dilution. Membranes were washed with TBST and incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, cat# 34080) according to the manufacturer’s instructions. Scanned Western blots were quantified using ImageJ software (RRID:SCR_003070), version 1.50a.

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Yan X., Tang B., Chen B., Shan Y, & Yang H. (2019). Replication Study: The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. eLife, 8, e43511.

Publication 2019

rabbit anti-CD44 mouse anti-ß-actin HRP-conjugated goat anti-rabbit HRP-conjugated horse anti-mouse SuperSignal West Pico Chemiluminescent Substrate

Actin Antibody Cd44 Cell Clone Dilution Goat Horse Membranes Mouse Protein Rabbit Tumor Western blots

Corresponding Organization :

Other organizations : Crown Bioscience (China), Science Exchange (United States), Center for Open Science

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Variable analysis

independent variables

Tumor samples

dependent variables

CD44 protein expression
β-actin protein expression

control variables

Total protein amount (40 µg) loaded on the membranes
Secondary antibodies used for detection (HRP-conjugated goat anti-rabbit and HRP-conjugated horse anti-mouse)

controls

Positive control: β-actin protein expression
Negative control: Not explicitly mentioned

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