Paxillin

V5-tagged WT PYK2, 4KR PYK2, and PYK2 Tyr402F were all transfected with and without MYC-SUMO1 in HeLa cells. V5-tagged WT PYK2 and 4KR PYK2 were also cotransfected with flag-PIAS1 and MYC-SUMO1. 4KR PYK2 and Tyr402F were generated using the QuikChange II site directed mutagenesis kit (Agilent Technologies, Wilmington, DE). Cells were rinsed with warm phosphate buffered saline 48 h after transfection and lysed with Kamiya buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm MgCl₂, 1 mm EDTA, 1% Triton-X-100). The lysate was incubated with anti-V5-agarose for two hours. Beads were washed three times with TBST then boiled with 2× LDS containing β-mercaptoethanol. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with antibodies to SRC (Cell Signaling, Danvers, MA) and paxillin (Santa Cruz, Dallas, TX). Phosphospecific antibodies for paxillin pTyr118 and SRC pTyr416 (Cell Signaling) were used to assess phosphorylation status.

Cells were lysed with Laemmli sample buffer containing 125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, β-mercaptoethanol, a protease inhibitor cocktail (Sigma), and a phosphatase inhibitor, PhosSTOP (Roche). The sample lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk and incubated with primary antibodies, TM311 (Sigma, T2780), E-cadherin (BD Transduction Laboratories, 610181), β-catenin (BD Transduction Laboratories, 610154), phospho-paxillin (Tyr 118) (Cell Signaling, 2541), paxillin (Santa Cruz, sc-5574), phospho-MLC2 (Ser19) (Cell Signaling, 3671), MLC2 (Santa Cruz, sc-15370), Myosin heavy chain IIA (Covance, PRB-440P), Myosin heavy chain IIB (Covance, PRB-445P), and β-actin (Sigma, A5441). The information of TM311, LC24, α/9d, and δ/9d (a synonym for WD4/9d) is described previously [15 (link)]. Primary antibodies were detected with HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research, 115-035-003) and goat anti-rabbit IgG (Jackson Immuno Research, 111-035-003). The intensity of bands was measured using Sigma ScanPro4.

Western blotting was performed to assess protein phosphorylation and expression levels in lung cancer cells treated with AC-93253 iodide as described previously [20 (link)]. The EGFR, STAT3 (F-2), PI3K, phospho-MEK1/2 (Ser 218/Ser 222), MEK, phospho-ERK (Tyr204), ERK, Paxillin, and p130cas were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phospho-Src (pY418), phospho-FAK (Tyr576), and FAK were purchased from Invitrogen (Carlsbad, CA, USA). Phospho-EGFR (Tyr1068), phospho-STAT3 (Tyr 705), phospho-PI3K (Tyr458), phospho-SAPK/Jun N-terminal kinase (JNK) (Thr183/Tyr185), SAPK/JNK, phospho-Paxillin (Tyr118), and phospho-p130cas (Tyr410) were purchased from Cell Signaling Technology (Beverly, MA, USA), and the primary antibody to Src was produced in our laboratory (ATCC CRL-2651). GAPDH (Upstate Biotechnology, Lake Placid, NY, USA) was used as a loading control. The mRNA expression levels of Src and related genes were detected using a real-time PCR machine (ABI prism 7300 Sequence Detection System, Applied Biosystems, Carlsbad, CA, USA) and SYBR Green Reagent (Roche, Basel, Switzerland). TATA-box binding protein (TBP) was used as an internal control (GenBank X54993). Details regarding the procedures and calculations used to perform the experiments are provided elsewhere [21 (link)].

Whole cell lysates from control (1 ×g) and hypergravity-exposed (20 ×g) cells were prepared with RIPA lysis buffer. Protein content was determined using a Protein Assay Kit (Thermo). Equal amounts of protein samples were resolved by reducing 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked for 2 h in 5% nonfat milk and incubated with primary antibodies against OPN (1:1000, Abcam), Runx2 (1:1000, Abcam), Paxillin (1:1000, Santa Cruz Biotechnology), p-FAK (1:1000, Cell Signaling), FAK (1:500, Santa Cruz Biotechnology), and GAPDH (1:2000, Santa Cruz Biotechnology) overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:10,000) for 2 h at room temperature. Lastly, the membranes were developed with enhanced chemiluminescence reagents (Millipore) and exposed to an X-ray film (Eastman-Kodak, Rochester, NY, USA).

Cell lysates were separated by SDS-PAGE in 8–10% gels and transferred into PVDF membranes. Antibodies used were: p-FAK^Y397 (611807), FAK (610088) (BD Transduction Laboratories, Lexington, KY); p-FAK (Tyr³⁹⁷) (sc-11765-R), cortactin (H-191), p-cortactin (Tyr⁴⁶⁶), paxillin (T-16), p-paxillin (Tyr¹¹⁸), E-cadherin (G-10), vimentin (E-5), actin (C-11) (Santa Cruz Biotechnology); α-Tubulin (T9026) (Sigma Aldrich); N-WASP (30D10) (Cell Signaling Technology); p-N-WASP (Ser^484/485) (Chemicon International); p-Arp2 (Thr237) (Biorbyt). Primary and secondary antibodies were incubated with the membranes using standard techniques. Immunodetection was accomplished using enhanced chemiluminescence and recorded with a quantitative digital imaging system (Chemidoc XRS with Image Lab, Bio-Rad, USA).

Antibodies used were: MIEN1 (Abnova, Taipei, Taiwan, and Life Technologies, Grand Island, NY), β-actin, ERK1, ERK2, ERK1/2, FAK, Paxillin, Tyr- 31 phosphorylated Paxillin (Santa Cruz Biotechnology, Dallas, TX), Ser-473 phosphorylated Akt, Akt, Tyr-416 phosphorylated Src, Src, Ser-3 phosphorylated Cofilin, Cofilin, Ser-536 phosphorylated NF-κB (Cell Signaling, Danvers, MA), Tyr-925 phosphorylated FAK (Life Technologies, Grand Island, NY), Tyr-397 phosphorylated FAK (Assay Biotech, Sunnyvale, CA). Alexa Fluor 488 phalloidin, Rhodamine phalloidin, Alexa Fluor 594 deoxyribonuclease I, Prolong Gold Antifade Mountant with DAPI were acquired from Molecular Probes (Life Technologies, Grand Island, NY). Human MIEN1 smart pool siRNA (L-014864-00) and control, non-targeting small interfering RNA pool (D-001810) were purchased from Dharmacon (Lafayette, CO).