Ab126061

The human NCKAP1 cDNA-containing plasmid was sourced from Shanghai Genechem Co., Ltd. (Shanghai, China). The plasmid was prepared using the pUC ori replicon. Cells were seeded in 6-well plates when the cell density was greater than 80%. Liposome 2000 was used for transfection and subsequently incubated in a 5% CO2 incubator. Transfection efficiency was assessed by Western Blot assay after 48 hours. Cellular proteins were extracted with RIPA lysate. The extracted proteins and markers were separated by electrophoresis with the help of SDS-PAGE gel (Western Protein Marker I: G2086, Servicebio, China). The protein blot was transferred onto a PVDF membrane (Microporous, USA). It was then blocked with 5% skimmed milk. Primary antibodies were incubated at 4 °C for 12 hours (NCKAP1 antibody: ab126061, GAPDH antibody: ab9485, Abcam, UK). The membranes were washed four times with TBST and incubated with a secondary antibody for 1.5 hours at room temperature (Goat Anti-Rabbit IgG H&L (HRP), Abcam, UK). The cell membranes were placed in Western LightningTM Chemiluminescent Reagent Chromogen (PerkinElmer, USA) for 30 seconds and then immediately placed in an exposure cassette, and finally scanned and imaged with an Epson Perfection V39 scanner, and then analyzed for the expression of each group of protein bands by Image-J software.

Lysates were collected on ice by scraping cells in RIPA buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1X protease and phosphatase inhibitors). The tubes were centrifuged for 10 min at 15,000 rpm and 4 °C. The lysate was transferred to a clean Eppendorf tube and measured using Precision Red.
The 40 μg of protein lysate was resolved on NuPAGE Novex 4–12% Bis-Tris gels and transferred onto a nitrocellulose membrane Bio-Rad system. Membranes were blocked with 5% BSA in TBS-T (10 mM Tris pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 20 min prior to overnight incubation with the primary antibody at 4 °C on a shaking incubator. Membranes were then washed three times for 5 min each in TBS-T. Membranes were then incubated at room temperature for 1 hour with secondary AlexaFluor conjugated antibodies, after which the blots were washed again for 5 min in TBS-T three times before being imaged on the Li-Cor Odyssey CLx machine. Images were then analysed using the Image Studio Lite Version 5.2. Lysates were harvested on 4 independent occasions from NCKAP1^fl/fl and NCKAP1^-/- MEFs.
Antibodies used: NCKAP1 (Abcam, Cambridge, UK: ab126061), CYFIP1 (Abcam: ab156016), WAVE2 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-33548) and α-Tubulin (Sigma, St. Louis, MO, USA; clone B512) as the loading control.