Phospho fak

bEnd.3 cells grown on 60-mm dishes treated with Au-NPs at prescribed periods, for example, 3~24 h for AQP1 protein analysis and 10~60 min for kinase phosphorylation analysis. After treatment, cell whole lysates were prepared as described [61 (link)]. Protein samples were separated by SDS-PAGE and electro transferred onto PVDF membranes. The membranes were blocked using TBST containing 5% skim milk, and incubated overnight at 4 °C with primary antibodies against AQP1, phospho-Akt, FAK (CUSABIO, Houston, TX), Cav1, phospho-Cav1, phospho-FAK (Abcam, Cambridge, UK), Akt, ERK, phospho-ERK (Santa Cruz, Dallas, TX), and β-actin (Sigma-Aldrich, Chicago, IL). The membranes were then washed in TBST and incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary IgG (1:5000). Finally, blots were developed using enhanced chemiluminescence (ECL), and images were captured and densitometrically analyzed using the BioSpectrum AC® Imaging system (UVP, Upland, CA).

Cells were lysed in RIPA buffer (Beyotime) containing protease inhibitor PMSF (Solarbio) at ratio (100:1 = RIPA: PMSF) and protein phosphatase inhibitor (Solarbio), then clarified by centrifugation at 13,000 g for 10 min at 4°C. Protein concentration was quantified using BCA Protein Assay Kit (Solarbio, Beijing, China). Western blot was performed by automatic protein analyser (Wes&Jess) according to the manufacturer's instruction. All protein levels were normalized to β‐actin. The blots were reacted with rabbit anti‐human against β‐actin (CST), phospho‐FAK, FAK, phospho‐MEK1, MEK1, phospho‐ERK1/2, ERK1/2, β3, αv, α5, β1 (all from Abcam).

Antibodies specific for the following proteins and tags were used: FAK (Abcam, #ab76496), ZDHHC5 (CST, #79842), β-actin (Proteintech, #81115–1-RR), Ubiquitin (CST, #20326), Flag (Abcam, #ab205606), Myc (Abcam, #ab9106), HA (CST, #3724), phospho-FAK (Abcam, #ab81298), Paxillin (Abcam, #ab32084), phospho-Paxillin (Abcam, #ab109547), AKT (Abcam, #ab8805), phospho-AKT (Abcam, #ab81283), ERK (Abcam, #ab184699), phospho-ERK (Abcam, #ab201015), α-Tubulin (Proteintech, #11,224–1-AP), ATP1A1 (Abcam, #ab7671), E-cadherin (Abcam, #ab219332), N-cadherin (Abcam, #ab18203), snail (Sigma-Aldrich, #SAB5700703), slug (Sigma-Aldrich, #SAB5700672), and β-catenin (Abcam, #ab32572).
The following reagents were used: MG132 (Beyotime, #S1748), cycloheximide (Sigma-Aldrich, #66–81-9), 2-bromopalmitate (Sigma-Aldrich, #238442), palmostatin B (Sigma-Aldrich, #178501), DMSO (Beyotime, #ST038), MeO-PEG-Mal (Sigma-Aldrich, #63187), hydroxylamine (Sigma-Aldrich, #159417), N-ethylmaleimide (NEM) (Sigma-Aldrich, #04260), and TCEP (Sigma-Aldrich, #646547).