Anti mouse igg horseradish peroxidase

Total protein extraction from EDL and pgWAT was performed as previously described (Milkiewicz et al., 2011 (link)). The following polyclonal primary antibodies were used: Ser473-pAkt, Akt, Ser563-pHSL, HSL and α/β-tubulin (Cell Signaling Technology, #4058, #9272, #4139, #4107 and #2148, respectively) and β-actin (sc-47778, Santa Cruz Biotechnology). Secondary antibodies were goat anti-rabbit or anti-mouse IgG-horseradish peroxidase (Jackson ImmunoResearch Laboratories, # 111-035-003, 115-035-003, respectively). Membranes were developed using enhanced chemiluminescence (SuperSignal^TM Westpico, #34080, ThermoFisher Scientific) and densitometry analysis was performed with ImageJ Analysis Software (NIH).

Indirect immunofluorescence was performed as previously described [5 (link)] by using the anti-EB1 antibody and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich). Cells were observed using a Leica DM-IRBE microscope, 100X magnification. All images were acquired using Metamoph software (Molecular Devices, Sunnyvale, CA) at identical acquisition settings, and were processed using Image J software. After cell lysis, 30 μg of total protein were loaded into a 12% SDS-PAGE gel. Anti-EB1 antibody, anti-α-tubulin and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch) were used. Chemiluminescence detection kit (Millipore) was used for visualization of protein bands. Chemiluminescent signal was acquired on a G:BOX imaging system (Syngene, Cambridge, UK) and quantification was done with Image J software.

Proteins were extracted from cell cultures and lysed with Laemmli sample buffer containing 2% SDS, 52.5 mM Tris-HCl and protease inhibitors (Roche diagnostics). Equal amounts of protein from each treatment were loaded into 12% SDS-polyacrylamide gels. Anti- EB1 (clone KT51, Abcam), anti-EB2 (Abcam), anti-EB3 (EPR11421(B), Abcam), anti-α-tubulin (Sigma-Aldrich), anti-acetylated tubulin (6-11B-1, Merck millipore), anti-detyrosinated tubulin (Abcam), and anti-GFP (Abcam), anti-GAPDH (Clone, source), anti-acetylated histone H3 (Merck millipore) and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch) were used. YL½ antibody (Merck Millipore) was used to detect both tyrosinated tubulin (~ 50 kDa) and tyrosinated EB1 (~ 30 kDa). U87 cells were transfected with GFP-EB1 and Detyrosinated-GFP-EB1 plasmids [19 (link)] using lipofectamineTM 2000 system (Invitrogen) and left to incubator for 24–48 h. Visualization of protein bands was performed with a chemiluminescence detection kit (Millipore) and the chemiluminescent signal was acquired with a G:BOX imaging system (Syngene). Quantification of western blot bands was performed with Image J software.