Anti mmp 1

Bowes cells, 2.5 × 10⁵ or 5 × 10⁵, were plated in 6- or 10-cm dishes, respectively, and incubated overnight. Cells were pelleted and lysed via sonication in cold cell extraction buffer (Invitrogen) 1X proteinase inhibitors (Calbiochem, Darmstadt, Germany) and 0.1 mM PMSF. Standard immunoblotting techniques were used (Huntington et al, 2004 (link); Croteau et al, 2013 (link)): 2–5 μg total protein was visualised with anti-pMEK (1 : 1000, Cell Signaling, Beverly, MA, USA), anti-Total MEK (1 : 1000, Cell Signaling), or anti-MMP-1 (1 : 5000, Millipore, Billerica, MA, USA), followed by the donkey anti-rabbit HRP secondary antibody at 1 : 5000 (Millipore). Signal was detected with Western Lightning Plus—ECL (PerkinElmer, Waltham, MA, USA).

After transfection with 859 siRNA and further 24 h incubation, cells were lysed in a mammalian cell lysis buffer (Clontech Laboratories, Inc.). Western blot analysis was then performed using conventional protocols. In brief, protein concentration was determined with Bradford assay (Bio-Rad) with bovine serum albumin as a standard (Sigma). Equal amounts of total protein were then separated on 12% polyacrylamide gels by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membrane. Antibodies and dilutions used in this study included anti-MMP1 (1:1000 dilution, Millipore, Billerica, MA, USA) and anti-GAPDH (1:2000 dilution, Millipore, Billerica, MA, USA). After being washed extensively, the membranes were incubated with goat anti-rabbit IgG peroxidase conjugate antibody (1:10000 dilution) for 1 h at room temperature and developed with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Membranes probed for hMMP1 were re-probed for GAPDH to normalize for loading and/or quantification errors and to allow comparisons of target protein expression or inhabitation to be made. Band density was measured by photoimage (Fusion-SL2-3500WL, Vilber Lourmat, France, www.vilber.com).

Confluent cultures of C4, C9 and VMM12 cells in 6-well plates were placed in 1 ml fresh DMEM/10% FBS for times as indicated for specific experiments. Total cell lysates were prepared with RIPA buffer (Sigma Aldrich, St. Louis, MO) and were separated on 10% SDS-PAGE gels (Bio-Rad, Hercules, CA). Serum-free conditioned medium (from 48 h cultures) was TCA precipitated, resuspended in 30 µl of 2× Laemmli buffer, and separated on 10% SDS-PAGE gels. Proteins were transferred to a PVDF membrane and visualized with anti-Col1A2 (1:200, Santa Cruz, CA), anti-MMP-1 (1:5000, Millipore), anti-pERK (9101), anti-total ERK (9102), anti-pAKT (4058), anti-total AKT (9272), anti-pSmad 2/3 (9520), anti-Smad 2/3 (5678), anti-total JNK (9252), anti-p-p38 MAPK (9215), or anti-p38 MAPK (8690) (1:1000 for each, Cell Signaling, Beverly, MA), anti-p-JNK (1:2000, Cell Signaling), or anti-Smad 7 (1:1000, Thermo Scientific, Rockford, IL), followed by the appropriate secondary antibody, donkey anti-rabbit HRP (1:20,000) or donkey anti-mouse (1:10,000). Signal was detected with Western Lightning Plus ECL (PerkinElmer, Waltham, MA, USA).