Rabbit anti human actin

Cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer, as described previously [42 (link)]. Cell lysates were centrifuged, and the protein concentration was determined by a Pierce bicinchoninic acid assay (BCA) protein kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of cellular protein were electrophoresed on 10–12% SDS–polyacrylamide gels and transferred to Hybond-Polyvinylidene fluoride (PVDF) () membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The primary antibodies used were rabbit anti-LC3 polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), mouse anti-adenovirus type 5 E1A (BD Pharmingen, San Diego, CA, USA), and rabbit anti-human actin (Sigma-Aldrich, St. Louis, MO, USA). Next, the membranes were incubated with anti-mouse immunoglobulin (Ig) or anti-rabbit Ig, peroxidase-linked, species-specific whole antibody (Thermo Fisher Scientific, Waltham, MA, USA). (Electrochemiluminescence) (ECL) reagents were used to detect the signals according to the manufacturer’s instructions (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The scanned band intensities were quantified using Gel-pro Analyzer 4.0 software (Media Cybernetics, Rockville, MD, USA) according to the manufacturer’s instructions. Densitometric values for each band were expressed as integrated optical density (I.O.D.) and were normalized to actin expression.

Cell extracts were prepared for WB as previously described [3 (link), 30 (link)]. Rabbit anti-LGR5 (1:1000, Santa Cruz biotechnologies), Mouse anti-LGR4 (1:400 Santa Cruz biotechnologies), rabbit anti-pPKC (1:1000, Cell Signaling Technology), rabbit anti-pJNK (1:1000, Cell Signaling Technologies) and rabbit anti-human actin (1:10,000, Sigma-Aldrich) were used as primary antibodies, whereas goat anti-rabbit IgG and goat anti-mouse IgG (1:10,000, Upstate Biotechnology, NY) were used as secondary antibodies.

Equal cell amounts of HCT116 from both CD74^+/+ and CD74^–/– genotypes were lysed with CelLytic M (Sigma) or RIPA (for nuclear proteins), and electrophoresed on 4%–20% TGX gels (Bio-Rad, Hercules, CA). Proteins were transferred to nitrocellulose and blocked with bovine serum albumin. Blots then were probed for proteins with the following antibodies: mouse anti-human CD74 (LN-2; Santa Cruz), rabbit anti-human actin (Sigma), rabbit anti-human pan-Akt (C67E7) and phospho-Akt (Ser473, D9E; Cell Signaling Technologies, Danvers, MA),²⁷ (link) rabbit anti-human ERK (137F5) and phospho-ERK (Thr202/Tyr204, D13.14.4E; Cell Signaling Technologies).