Erk 9102

Specific antibodies against phospho-p38 (#9211), phospho-JNK (#9251), phospho-ERK (#9106), phospho-MEK1/2 (#9154), and ERK (#9102) were obtained from Cell Signaling Technology (Danvers, MA). Monoclonal anti-β-actin (A5441) was obtained from Sigma—Aldrich (St. Louis, MO). Recombinant mouse M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN). KP-A159, 8-phenyl-2-(phenylthio)-6,7-dihydro-5H-cyclopenta[b]thiazolo[5,4-e]pyridine (Fig 1), is the compound from an in-house chemical library and was synthesized as previously described [16 (link)].

Target cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor (RIPA:PMSF = 100:1). After determining the protein concentration by the Bicinchoninic Acid (BCA) method, protein samples (40 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 h with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin (BSA). The membranes were then incubated on a shaking bed with primary antibodies (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab106077, Twist ab50887, Zeb 2 ab138222, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT ab38449, mTOR ab32028, p-mTOR ab109268, Abcam; p-ERK (Thr202/Tyr204) #4370, ERK #9102, Cell Signaling), at a 1:2,000 dilution overnight at 4 °C. The next day, the membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Finally, positive labeling of the proteins on the membranes was visualized by enhanced chemiluminescence (ECL) using a Bio-Rad ECL kit (Solarbio).

Primary antibodies were diluted as follows: β-Actin (C-4, 47778, Santa Cruz) 1:2000; P-CDK1 (Y15)(9111, Cell Signaling) 1:1000; T-CDK1 (77055 and 9112, Cell Signaling) 1:1000; P-CHK1 (S345)(2348, Cell Signaling) 1:1000; T-CHK1 (G-4, 8408, Santa Cruz) 1:1000; P-ERK (9106, Cell Signaling) 1:1000; ERK (9102, Cell Signaling) 1:1000; Phospho-Histone H2A.X (Ser139)(γH2AX)(2577, Cell Signaling) 1:1000; H2A.X (2595, Cell Signaling) 1:1000; PARP (9542, Cell Signaling) 1:1000; Ras (sc-30, Santa Cruz) 1:1000; P-RSK (S380)(9341, Cell Signaling) 1:1000; T-RSK (601225, BD Biosciences) 1:1000; TIMELESS (A300-961A, Bethyl) 1:5000; and α-tubulin (B-5-1-2, Santa Cruz) 1:2500.

Cells were harvested using radio-immunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Bimake, Houston, TX). Equal amounts of protein were loaded per lane into an SDS-PAGE followed by transferring onto a nitrocellulose membrane (BioRad). The blots were blocked with 5% nonfat dried milk followed by incubation in the respective antibodies. After several washes, membranes were incubated with appropriate secondary antibodies and imaged using either chemiluminescence or the LICOR Odyssey Infrared Imaging System (Lincoln, NE). Antibodies for western blot analysis were used at 1:1000 dilution unless specified otherwise. HA-Tag #sc-7392, Fra-1 #sc-183, ATF-2 #sc-187, GAPDH #sc-137179 were from Santa Cruz Biotechnology; anti-phosphor-MK2 #07-155 from Upstate Biotechnology; Myc-tag #2866, ERK #9102, phosphor-ERK #4376, p38^MAPK #9212, phosphor-p38^MAPK #4511, JUN #9165, MK2 #3042, Jab1 #9444, Cyclin D1 #2922, βActin #4967 were from Cell Signaling; FLAG #F3165 was from MilliporeSigma; uPA #395 and uPAR #3937 were from American Diagnostica used at 1:500 dilution; Anti-phosphorylated JAB1 (Ser¹⁷⁷) pAb was commercially prepared by EZ Biolab (Carmel, IN) using synthesized AVVIDPTRTI(pS)AGKVN peptide and used at 1:500 dilution. All blots derive from the same experiment and were processed in parallel.