Phospho fak tyr397

Cell lysates (20–40 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes using primary antibodies for ASPH (FB50, homemade); MMP14 (#13130S), SRC (#2109S), SRCY416 (#6943S), SRCY527 (#2105S), FAK (#13009S), Phospho-FAK Tyr397 (#8556S), FAK Tyr566/567 (#3281S), FAK Tyr925 (#3284S), and Alexa Fluor® 488 Conjugate (#5198S) from Cell Signaling Technology; MMP1 (sc-58377), ADAM12 (sc-25579), and ADAM15 (sc-16530) from Santa Cruz Biotechnology. Protein bands were visualized by ChemiDoc™ Touch Imaging System (Bio-Rad).

The following antibodies were used: PR65 (sc-15355, Santa Cruz, CA, USA), PP2Ac (05-421, Millipore), FLAG M2 (F1804, Sigma Aldrich), HA (sc-7392, Santa Cruz), phospho-AKT (Thr308) (9275, Cell Signaling), phospho-AKT (Ser473) (9271, Cell Signaling), phospho-SRC (Tyr416) (6943, Cell Signaling), phospho-FAK (Tyr397) (3283, Cell Signaling), phospho-PP2Ac (Tyr307) (ab32104, Abcam), SRC (2108, Cell Signaling), p21 (2946, Cell Signaling), β-actin (sc-4778, Santa Cruz), phospho-SAPK/JNK (Thr183, Tyr185) (9251, Cell Signaling), phospho-c-Jun (Ser73) (9164, Cell Signaling), phospho-c-Jun (Ser63) (9261, Cell Signaling), and c-Jun (sc-1694, Santa Cruz).

The human chondrocyte cell line C28/I2 was used^{68 (link)}. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) containing 10% FBS (Hyclone) and 1% penicillin/streptomycin (Lonza), and maintained at 37 °C and 5% CO₂ in a humidified incubator. Neon transfection system (Invitrogen) was used to transfect the biosensors into the cells. After transfection, two parts 3% agarose gel solution was mixed with one part 3 × DMEM containing transfected cells to produce the cell/gel construct with 2% agarose and 1 × DMEM. The mixture was injected into the μ-slide flow chamber (Ibidi) and cooled at room temperature for 30 min to allow gelation. The cell/gel construct was incubated in DMEM containing 0.5% FBS for 24 h before imaging 7 experiments. For Western blot analysis, cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer. Isolated proteins were fractionated using 10% SDS gels and electro-transferred to Immobilon-P membranes. The membrane was incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling). The primary antibodies used were Pyk2 (Cell Signaling), phospho-Pyk2 (Tyr402) (Cell Signaling), FAK (Cell Signaling), phospho-FAK (Tyr397) (Cell Signaling), and β-actin (Sigma). Signal intensities were quantified by a luminescent image analyzer (Fujifilm).

Sources of chemicals are as follows: NDGA, caffeic acid (Sigma, USA); AG538 (Cayman, USA); picropodophyllin (PPP, Selleck, USA), fibronectin protein (FN, Santa Cruz, USA). Antibodies against NRP1 (diluted 1:1000), FN (diluted1:1000), β-actin (diluted 1:10000) and GAPDH (diluted 1:1000) were purchased from Abcam, USA. Antibodies against total FAK (diluted 1:1000), phospho-FAK (Tyr397) (diluted 1:1000), DyLight 800-conjugated secondary antibodies (diluted 1:10000), DyLight 488-conjugated secondary antibodies (diluted 1:1000) and HRP-conjugated secondary antibodies (diluted 1:1000) were purchased from Cell Signaling Technology, USA.

RPE samples were obtained from the eyes of control and KO mice at ZT 23, ZT 1, and ZT 3 using the methodology described in Baba et al., (2010) and then lysed in ice-cold RIPA buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1.0% Nonidet −40; and 1.0% sodium deoxycholate), 1x protease inhibitors, and 1x phosphatase inhibitor I and II. Following lysis and separation on SDS/PAGE gel, the proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Trans-Blot Turbo transfer system; Biorad Laboratories, Hercules, CA, USA; #1704156). The blot was incubated overnight at 4°C with Phospho-FAK (Tyr 397; 1:1000; Cell Signaling, Danvers, MA, USA; #3283), FAK (Cell Signaling; #3285). RPE-65 (1:2500, generous gift of T.M. Redmond, NEI). RPE-65 was used as a loading control for the amount of RPE protein present in the samples. Then, they were incubated with anti-rabbit horseradish peroxide (HRP; 1: 10000; Cell Signaling; #7074S) and developed. Band intensities were quantified by densitometry using Image J (1.51w).