Lamemmli buffer

Whole cell lysates were generated using lamemmli buffer (Bio-rad) in the presence of proteinase inhibitor cocktail. Lysates were run on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were cut at approximately 60 kDa; the top portion of the membrane was used to probe for cyclin T1 (CycT1) (75 kDa), the bottom portion was used to probe for CDK9 (40 kDa), Hexim 1 (Hex1) (54 kDa) and βactin (55 kDa). Membranes were blocked in 5% non-fat milk (NFM) for at least 1 hour and blotted overnight with rabbit anti-human cyclin T1 antibody (Santa Cruz Biotechnology), rabbit anti-human CDK9 (Santa Cruz Biotechnology), rabbit anti-human Hex1 (Santa Cruz Biotechnology), and rabbit anti-human β actin (abcam) in 5% NFM. Membranes were washed 3 x in PBS with 0.05% Tween 20, and then blotted for 1 hour with HRP anti-rabbit IgG, in 5% NFM. After washing 3 x with PBS with 0.05% Tween 20; membranes were treated with ECL Plus chemiluminescence reagent (Promega) for 5 minutes and imaged using Odyssey Fc imaging system and Image Studio software (LI-COR). Reprobed membranes were stripped with NewBlot Stripping Buffer (LI-COR) then washed 3 x with PBS.

Whole cell lysates were generated using lamemmli buffer (Bio-rad) in the presence of proteinase inhibitor cocktail. Lysates were run on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were cut at approximately 60 kDa; the top portion of the membrane was used to probe for CycT1 (75 kDa) and the bottom portion was used to probe for phospho-(p-) (42 kDa) and total IKBα (40 kDa), p- and total ERK (42 kDa), and β-actin (55 kDa). Membranes were blocked in 5% non-fat milk (NFM) for at least 1 hour and blotted overnight with mouse anti-human cyclin T1 antibody (sc-271348, Santa Cruz Biotechnology), mouse anti-human p-ERK (P-p44/42 MAPK) (9106S, Cell Signaling), rabbit anti-human ERK (p44/42 MAPK) (9102S, Cell Signaling), mouse anti-human p-IKBα (9246S, Cell Signaling), mouse anti-human IKBα (48145S, Cell Signaling) and rabbit anti-human β-actin (ab8227, abcam) antibodies in 5% NFM. Membranes were washed 3 x with PBS with 0.05% Tween 20, and then blotted for 1 hour with HRP anti-rabbit IgG, in 5% NFM. After washing 3 x with PBS with 0.05% Tween 20; membranes were treated with ECL Plus chemiluminescence reagent (Promega) for 5 minutes and imaged using Odyssey Fc imaging system and Image Studio software (LI-COR). Reprobed membranes were stripped with NewBlot Stripping Buffer (LI-COR) then washed 3 x with PBS.

Electrophoresis was carried out in a vertical Bio-Rad Mini-Protean^® system using a Tris/Glycine/SDS buffer (Bio-Rad Cat# 161-0772; Hercules, CA, USA). The sample was diluted 1:1 with a Lamemmli buffer solution containing 950 μL Lamemmli buffer and 50 μL β-mercaptoethanol Lamemmli buffer (Bio-Rad Cat# 161-0737; Hercules, CA, USA)/β-mercaptoethanol (Fisher Scientific Cat# O3446I-100; Pittsburgh, PA, USA). The mixture was heated for 5 min at 99 °C. Proteins were separated using 4%–15% gradient Mini-Protean^® TGX™ 30 μL well precast gels (Bio-Rad Cat# 456-1083; Hercules, CA, USA). Gels were loaded with 30 μL of sample and run at 155 V for 45 min. Gels were placed on a shaker set to low speed (25 RPM) and stained using 50 mL of Acqua Stain (Bulldog Bio Co. Cat# AS001000; Portsmouth, NH, USA) for 60 min. The bands were photographed with a PowerShot G10 Digital Canon Camera (Tokyo, Japan) equipped with an orange filter. A 250–10 kDa protein ladder (Dual Color Precision Plus Protein Standards. Bio-Rad Cat# 161-0374; Hercules, CA, USA) used was as a standard.