Mouse anti myc antibody

For fluorescence-based measurements of the ratio of surface-to-total AQP-5, HSG cells transfected with pCMV6-AQP-5-Myc construct were incubated for 16 hrs. After pretreatment for indicated time with 100 μM ZnCl₂, cells were fixed in PBS containing 4% formaldehyde and stained for surface AQP-5 using goat anti-AQP-5 antibody (1:100, Santa Cruz) in PBS under a non-permeable condition overnight at 4 °C. Cells were washed three times with PBS, permeabilized in PBS containing 0.5% Triton X-100 for 10 min, and stained for total Myc-tagged AQP-5 using mouse anti-Myc antibody (1:100, Cell Signaling) for 1 hr at room temperature and then a Cy3-conjugated anti-goat secondary and Alexa Flour 488 or 647-conjugated anti-mouse secondary antibody (1:500, Jackson ImmunoResearch Laboratories) for 30 min. Images were acquired with an LSM 700 laser-scanning confocal microscope (Carl Zeiss) using a C-Apo 40 × 1.20 W objective lens. Cells were outlined, and mean fluorescence intensity was measured for each channel using ZEN imaging software (Carl Zeiss). For quantification of surface/total AQP-5 level, the fluorescence intensity of surface AQP-5 was divided by that of total AQP-5. The ratio of surface-to-total AQP-5 fluorescence intensity was compared with that of vehicle-treated controls

The following antibodies were used: mouse anti-Myc antibody (Cell Signaling, Cat. #2276#, 1:8000 for western blots and 1:5000 for confocal imaging); rabbit anti-GFP antibody (Abcam, ab290, 1:5000); guinea pig anti-p62 antibody (Progen, Cat. #Gp62-C#, 1:5000); rabbit anti-actin antibody (Sigma, Cat. #A2066#, 1:1000); Alexa Fluor® 647-conjugated goat anti-mouse IgG (A21236, 1:1000); HRP-conjugated goat anti-mouse IgG (1:5000); goat anti-rabbit IgG (1:5000); goat anti-guinea pig IgG (1:5000).

HeLa cells were cultured in six-well plates at a density of 1×10⁵ cells/ml, and after 24 h the cells were transfected with WT-RANKmyc, W434X-RANKmyc or G280X-RANKmyc using the JetPRIME transfection reagent. After 48 h, the cells were prepared in a radioimmunoprecipitation assay buffer (RIPA; deoxycholic acid (12.5 mM), SDS (3.5 mM) and IGEPAL (1%) in PBS) containing 1% protease inhibitor cocktail for western blot analysis as described previously (Crockett et al. 2011 (link)). The proteins on the PVDF membrane were probed with mouse anti-RANK antibody (Imgenex, clone 9A725, #IMG-128A) followed by donkey anti-rabbit-800 secondary antibody (LI-COR Biosciences, Cambridge, UK) and then with mouse anti-Myc antibody (Cell Signaling Technology, Danvers, MA, USA) followed by donkey anti-mouse-680 secondary antibody (LI-COR Biosciences). The blots were analysed on an LI-COR Infrared imager with Odyssey analysis software.

Whole cell extracts were lysed on ice in RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.4, 1% NP-40, 0.1% SDS), supplemented with complete EDTA-free protease inhibitor cocktail (Roche). Total protein concentrations were quantified using the BCA protein kit (Life Technologies), and cell lysates containing 20 μg of protein were subjected to electrophoretic separation on denaturing polyacrylamide gels under reducing conditions, followed by transfer to PVDF membranes. The latter were then probed with a mouse anti-myc antibody (1:1000, Cell Signalling Technologies), a rat anti-MLKL antibody (1:2000) [15 (link)] and a rabbit anti-GAPDH antibody (1:2500, Trevigen), followed by the appropriate secondary horseradish peroxidase-conjugated antibody (1:3000, Cell Signalling Technologies). The signal was visualized using the chemiluminescent ECL reagent (Life Technologies).