Anti vinculin

Cells were harvested and lysed in Pierce IP Lysis Buffer (Thermo Fisher Scientific) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). After centrifugation of the lysates, the protein concentrations of the supernatants were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The lysates were boiled for 5 min with Sample Buffer Solution with 3-mercapto-1, 2-propanediol (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). The samples were subjected to 5%–20% gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Once separated, the proteins were electrophoretically transferred to polyvinylidene fluoride polymer membranes, which were incubated with primary antibodies against Rictor (#2114), Raptor (#2280), Mcl-1 (#5453), Bcl-xL (#2762), Bcl-2 (#2870), XIAP (#2042), cFLIP (#56343), cIAP1 (#7065), cIAP2 (#3130), AKT (#9272) (Cell Signalling Technology, Danvers, MA, USA) and anti-vinculin (#CP74; Merck Millipore), and then incubated with species-specific HRP-conjugated secondary antibodies. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was used for protein detection and images were captured digitally using the ImageQuant LAS4000 camera system (GE Healthcare).

Whole cell lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were resolved on 10–15% SDS-PAGE gels and electroblotted onto PVDF membrane. Membranes were blocked with 5% BSA or dried skimmed milk in TBS-T and incubated in primary antibodies in blocking buffer: anti-pRPA32-Ser³³ (A300-246A Bethyl Laboratories), anti-phospho-γH2AX-Ser¹³⁹ (Merck Millipore, 05–636), anti-pCHK1-Ser³⁴⁵ (Cell signalling #2348), anti-RNA polymerase II CTD repeat YSPTSPS (phospho-S5) (Abcam ab5401), anti-PUM2 (Bethyl Laboratories A300-202A), anti-RNASEH1 (SantaCruz Biotechnologies, H-4, sc-376,326), anti-β-actin (Abcam ab6276), anti-Vinculin (Merck Millipore, V9131), anti-GAPDH (Cell Signalling 14C10 #2118). Membranes were washed and incubated with HRP-conjugated secondary antibodies and blots developed with Luminata™ forte (Merck-Millipore) and imaged using iBright (Invitrogen).

The APOPercentage™ kit was acquired from Biocolor Ltd. (Carrickfergus, Ireland); the Caspase-Glo^®-3/7 assay kit, MitoToxGlo^™, and MultiTox-Glo multiplex kit were acquired from Promega (Madison, WI, USA); the Topoisomerase-I relaxation assay kit was acquired from TopoGen (Buena Vista, CO, USA); the Annexin V-FITC apoptosis kit was acquired from BD Pharmingen (San Diego, CA, USA); the 5- (and 6) -chloromethyl-2′,7′-dichlorofluorescein diacetate acetyl ester (CM-H₂DCFDA) and MitoTracker Red (CMXRos) were acquired from Molecular Probes (Invitrogen, OR, USA); the DAPI (4′,6-diamidino-2-phenylindole), TRITC-conjugated phalloidin, and anti-vinculin were acquired from Merck Millipore (Darmstadt, Germany); Hank’s balanced salt solution (HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM), and phosphate buffered saline (PBS) were acquired from Gibco (USA); penicillin/streptomycin, Triton X-100, Tween-20, and paraformaldehyde were acquired from Sigma-Aldrich (St. Louis, MO, USA); and NucBlue Live ReadyProbes^TM Reagent was purchased from Thermo Fischer (Johannesburg, South Africa). All other chemicals and reagents used in this study were of analytical grade.

Primary antibodies used in Western blot analysis include anti-GST (own antibody); anti-GFP (1:1,000; #PA1-980; Thermo Fisher Scientific); anti-Flag (1:1,000; #F742 and #F3165; both from Merck); anti-C-RAF-N-terminal (1:1,000; #ab181115; Abcam); anti-C-RAF-pS259 (1:1,000; #ab173539; Abcam), anti-C-RAF-pY340/341 (1:1,000; #sc-16806; Santa Cruz Biotechnology); anti-vinculin (1:1,000; #V9131; Merck); anti-SIRT4 (1:1,000; #69786; Cell Signaling); anti-ERK(1/2) (1:1,000; #9102; Cell Signaling); anti-p-ERK(1/2) (1:1,000; #4370; Cell Signaling); and anti-KRAS (1:1,000; 11H35L14; Thermo Fisher Scientific). Secondary antibodies employed were from LI-COR (anti-mouse 700 nm: IRDye #926-32213; anti-rabbit 800 nm: IRDye #926-6807).

Mesoporous silica nanoparticles (particle size 200 nm, pore size 4 nm), Poly(l-Lactide) (Mn 5000), N-hydroxysuccinimide (NHS, 98%) and N-(3-dimethylaminopropyl)-N′-ethylcarbo-diimide (EDC, > 97%), Xanthine oxidase (XO, ≥ 0.4 units/mg protein) and 2-amino-4-hydroxypteridine Pterine (PR) were purchased from Sigma (St. Louis, MO). RSV was purchased from Calbiochem^® (EMD Millipore, USA). The FITC-labeled LDL peptide ligand was synthesized by Bankpeptide Inc (Hefei, China) according to the previous report [16 (link)]. The sequence of the peptide is Acp-(Cys-Met-Pro-Arg-Leu-Arg-Gly-Cys)c-NH₂. The N-terminal of peptide was labeled by green fluorescent FTIC, and the C-terminal was amidated.
The primary antibodies, anti-Vinculin and anti-Iba1 mouse monoclonal antibody, anti-Myosin, anti-iNOS, and anti-NADPH p47^phox rabbit polyclonal antibody were purchased from EMD Millipore (Billerica, MA, USA). The anti-occludin rabbit polyclonal antibody was purchased from Lifespan Biosciences, Inc. (Seattle, WA, USA).

For the determination of protein expression, cells were lysed in RIPA buffer supplemented with a cocktail of protease inhibitors (cOmplete™, Mini, EDTA-free, Roche, Basel, Switzerland). Western Blot analysis was performed, as previously described [27 (link)]. Briefly, 20 µg of proteins was separated on SDS-PAGE (Bolt™ 4–12% Bis-Tris mini protein gel, Thermo Fisher Scientific) and electrophoretically transferred to PVDF membranes (Immobilon-P membrane, Merck). Membranes were incubated for 1 h at RT in Tris-buffered saline solution (TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 5% non-fat dried milk, then incubated overnight at 4 °C in TBST (TBS + 0.5% Tween) supplemented with 5% BSA and anti-ICAM-1, anti-VCAM-1, or anti-tissue factor (TF) purified rabbit polyclonal antibodies or anti-CD31/PECAM mouse monoclonal antibody (1:2000, Cell Signaling Technology, Beverly, MA, USA). Anti-vinculin (Merck) or anti-β-actin (Cell Signaling Technology) mouse monoclonal antibodies (1:2000) were employed as the internal standard. Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with the iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with the iBright Analysis Software.

The following antibodies were used in this study: anti-HOIL-1L (clone 2E2; Merck MABC576), anti-HOIP (Merck SAB2102031), anti-IκBα (44D4; Cell Signaling Technology 4812), anti-Ser32/36-phospho-IκBα (5A5; Cell Signaling Technology 9246), anti-SHARPIN (NOVUS Biologicals NBP2-04116), anti-ubiquitin (P4D1; Santa Cruz Biotechnology sc-8017), anti-vinculin (Merck V9131). All antibodies were diluted in TBS 5% (w/v) BSA, 0.05% (v/v) Triton according to the manufacturer’s recommended dilutions.