Mouse monoclonal anti tubulin t5168

Primary antibodies used in this work were rabbit polyclonal anti-HA (ab9110) from Abcam (Cambridge, UK); mouse monoclonal anti-RAB7A (sc376362), anti-vimentin (sc6260), anti-p-αPAK (sc-135755), anti-αPAK (sc-166887), anti-cofilin-1 (sc-53934), anti-caspase 9 (sc-56073), rabbit polyclonal anti-myc (sc-789), anti-p-AKT 1/2/3 (sc7985-R), anti-AKT 1/2/3 (sc8312), anti-p-PKA α/β/γ cat (sc32968), anti-PKA (sc903), and ROCK-2 (sc5561), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-β-catenin (610154) from BD Transduction Laboratories (San Jose, CA, USA); rabbit polyclonal anti-cleaved caspase 9 (#7237), anti-NF-kB p65 (#8242), anti-matrix metalloproteinase 2 (MMP2) (#4022), anti-p-vimentin S38 (#13614), and anti-p-p65/RelA (Ser536) (#3033) from Cell Signaling Technology (Leiden, The Netherlands); mouse monoclonal anti-tubulin (T5168) from Sigma-Aldrich and rabbit polyclonal p-ROCK2 (GTX122651) from GeneTex (Irvine, CA, USA). Secondary antibodies conjugated to fluorochromes (for immunofluorescence analyses) or horseradish peroxidase (HRP, for immunoblot analyses) were from Invitrogen (Carlsbad, CA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Crowded plates of various C. elegans strains were harvested and washed twice in M9 and then lysed using the following lysis buffer: 25 mM Tris (pH 7.5), 300 mM NaCl, 1% triton X-100 and 1X protease inhibitor. 25 μg of total protein was loaded in each lane. For transgenic animals, 25 worms expressing GFP (used as a transformation marker) were picked, boiled in sample buffer (4x Laemmli sample buffer, BIO-RAD) and loaded on an SDS gel. For detection of protein the nitrocellulose membranes (GE Health care) were blocked with 5% skimmed milk (Blotting-Grade Blocker, BIO-RAD) diluted in PBS-T. Antibody dilutions (primary antibody: rabbit monoclonal anti-HA antibody (C29F4; Cell Signaling Technology) 1:5000, mouse monoclonal anti-TUBULIN (T5168; SIGMA) and secondary antibodies: swine anti-rabbit HRP (1:3000, Dako) or goat anti-mouse HRP (1:3000, Dako)) was done in 5% skimmed milk in TBST and washes were carried out in PBS-T. Detection of the bound antibodies was performed using an ECL detection kit (Immobilon Western; Millipore) and visualized with a digital camera (VersaDoc; Bio-Rad).