Rabbit polyclonal antibody against human sllp1

For the analysis of cytoplasmic SLLP1 protein expression, myeloma cell lines were fixed using FACS Lysing Solution, followed by permeabilization with Permeabilizing Solution (both from BD Biosciences). Cells were stained with a rabbit polyclonal antibody against human SLLP1 (Sigma) or an appropriate isotype control antibody followed by incubation with a secondary FITC-conjugated goat anti-rabbit IgG antibody from Jackson ImmunoResearch (Suffolk, UK). Samples were analyzed using a FACSCalibur cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star, Ashland, OR, USA).

Whole cell protein extracts were prepared in RIPA buffer containing a cocktail of protease inhibitors (Sigma, Steinheim, Germany). Testis lysate used as a positive control was obtained from Abnova (Taipei, Taiwan). 293 cells were transfected with an SLLP1 expression plasmid (Origene, Rockville, MD) using Lipofectamine 2000 (Lifetechnologies) and harvested after 3 days. Protein concentrations were determined by BCA assay (Thermo Scientific) and immunoblot analysis was performed as previously described [13 (link)] applying 80 µg of protein per lane. The primary antibodies were a rabbit polyclonal antibody against human SLLP1 (Sigma) used at a dilution of 1:1,000 and a mouse anti-human monoclonal antibody against β-actin (ACTB; Cell Signaling Technology, Danvers, MA) used at a dilution of 1:3,000. Secondary antibodies were an HRP-labeled anti-rabbit monoclonal antibody (R&D Systems, Minneapolis, MN, USA) used at a dilution of 1:2,000 or an HRP-labeled anti-mouse monoclonal antibody (R&D Systems, Minneapolis, MN, USA) used at a dilution of 1:3,000, respectively. Specific antibody binding was visualized by chemiluminescence (PerkinElmer, Waltham, MA, USA).