Anti rat 7077

Total cell extracts (TCEs) were prepared in denaturing buffer (50 mM Tris–HCl pH 8.0, 600 mM NaCl, 0.5% sodium deoxycholate, 1% NP40 0.1% sodium dodecyl sulfate, and 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). Proteins were resolved using precast Bolt Novex Bis–Tris Gels 4–12% (Life Technologies), transferred to nitrocellulose membranes (Bio-Rad), and immunoreactivity was determined using ECL (Amersham). Acquisition and densitometric analysis of images were obtained using Image Lab software (Bio-Rad). Employed Abs were as follows: anti-HIPK2 (rat monoclonal Ab C5C6 kindly provided by M. L. Schmitz); anti-Spastin (1:100; #sc-53443), anti-GAPDH (1:1,000; #sc-32233), anti–α-tubulin (1:1,000; #sc-5286), anti-actin (1:1,000; #sc-47778), anti-ALIX (1:200; #sc-53538), anti-p62 (1:1,000; #28359), anti-lamina A/C (1:1,000; #sc-20680) by Santa Cruz Technology; anti-CEP55 (1:1,000; #00055165-A01), and anti-NBR1 (1:500; #H00004077-B01P) by Abnova; anti-TSG101 (1:500: #ab30871) and anti-H2B (1:1,000; #ab52484) by Abcam; anti-LC3 (1:1,000; #L8918 by Sigma–Aldrich) recognizing both the cytosolic LC3-I and lipidated LC3-II forms of LC3. Anti–horseradish peroxidase–conjugated goat anti–mouse #7076, anti–rabbit #7074, and anti–rat #7077 (Cell Signaling Technology).

The MEK inhibitor U0126 and the Rac1 inhibitor NSC23766 were purchased from Calbiochem/Merck. GB88 and GB110 were a kind gift of Dr. David B. Fairlie (The University of Queensland, Brisbane, Australia) [33 (link),34 (link)]. The PAR2-selective peptide agonist SLIGKV-NH₂ and the PAR1-selective agonist peptide TFLLRN-NH₂ (STRAP-1) were obtained from Bachem (Bubendorf, Switzerland). Synthesis, coupling, cleavage from the resin and characterization of the PAR2-selective peptide 2-furoyl-LIGRLO-NH₂ (2f-LI, EC₅₀ = 2.5 μM) were done as described in detail before [26 (link)]. The following primary antibodies were used: anti-ALK5 antibody (TGFβ RI (V22), Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-ERK1/2 (#4370, Cell Signalling Technology, Frankfurt/Main, Germany), anti-HSP90 (both #sc-7947 and #sc-13119), anti-ERK1/2 (#AF1576, R&D Systems, Wiesbaden, Germany), and anti-HA (clone 12CA5, Roche, Mannheim, Germany). HRP-linked anti-rabbit (#7074), anti-mouse (#7076), and anti-rat (#7077) secondary antibodies were from Cell Signaling Technology. The rhTGF-β1 (#300-023) was purchased from ReliaTech (Wolfenbüttel, Germany) and used at a concentration of 5 ng/mL.

The following primary antibodies were used: Anti-phospho-ERK1/2 (#4370, Cell Signalling Technology, Frankfurt/Main, Germany), anti-HSP90 (both #sc-7947 and #sc-13119), anti-ERK1/2 (#AF1576) and phospho-Smad3(Ser423/425) (#AB3226), both from R&D Systems, Wiesbaden, Germany anti-Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), anti-Rac1 (#610650), anti-CIP1/WAF1 (#610233) (both from BD Biosciences, Heidelberg, Germany), anti-proteasome (#PW-8195, Biomol, Plymouth Meeting, PA, USA), anti-HSP27 (#SA-348, Biomol), anti-MnSOD (#06-984, Upstate Cell Signaling Solutions, Lake Placid, NY, USA), anti-osgin (#H00029948-B01P, Abnova, Taipei, Taiwan), anti-β-actin (#A1978, Sigma-Aldrich, Deisenhofen, Germany). HRP-linked anti-rabbit (#7074), anti-mouse (#7076) and anti-rat (#7077) secondary antibodies were from Cell Signaling Technology, anti-goat secondary antibody (#ab6741) was from Abcam (Cambridge, UK). The rhTGF-β1 (#300-023) was purchased from ReliaTech (Wolfenbüttel, Germany) and used at a concentration of 5 ng/mL for the breast cancer cell lines and 10 ng/mL for HMEC.