K562 cells were lysed with mammalian cell lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) containing protease and phosphatase (both Nanjing KeyGen Biotech Co., Ltd.) inhibitors. The BCA protein determination method was used to detect the amount of protein. The total protein content was extracted with a 10% Tris-HCl gradient gel (Bio-Rad Laboratories, Inc.) and transferred onto PVDF membranes, which was blocked using 5% non-fat milk in TBS/Tween-20 (0.1%) for 2 h at room temperature. The membrane was then probed with antibodies for GATA-1 (monoclonal rabbit anti-human; 1:10,000; cat. no. ab181544; Abcam), SMAD2 (monoclonal rabbit anti-human; 1:10,000; cat. no. ab40855; Abcam) and α-tubulin (monoclonal mouse anti-human; 1:10,000; cat. no. ab7291; Abcam) and incubated overnight at 4°C. The following day, the PVDF membrane was taken out of the refrigerator, reheated at room temperature for 1 h and washed in TBS/Tween-20 (0.1%). The secondary antibodies (Goat anti-mouse IgG, HRP conjugate, cat. no. SA00001-1; Goat Anti-Rabbit IgG, HRP conjugate, cat. no. SA00001-2; ProteinTech Group, Inc.) were added and incubated at room temperature for 1 h, followed by washing with TBS/Tween-20 (0.1%). The visualization reagent (cat. no. 34094; Thermo Fisher Scientific, Inc.) was prepared according to the instructions of the kit. Quantity One software (4.6.2; Bio-Rad Laboratories, Inc.) was used for density analysis of protein bands.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.