Pan actin rabbit polyclonal

Cells were incubated in lysis buffer (1 % Triton X-100, 50 mM HEPES, 150 mM NaCl, 1 mM MgCl_2, 1 mM EGTA, 10 % glycerol, and protease inhibitor cocktail) for 1 h at 4 °C. Cell lysates were harvested by scraping and centrifuged at 14,000 rpm for 10 min at 4 °C. Normalized samples (using the BCA assay (Fisher Scientific, Pittsburgh, PA)) were run on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes for western blotting. Bound antibody was detected using enhanced chemiluminescence reagent followed by exposure to film. Primary antibodies were used at the following dilutions and obtained from the following sources: Ago2 rabbit monoclonal (#2897, 1:500), caspase 2 mouse monoclonal (1:1000), caspase 3 rabbit monoclonal (1:1000), caspase 8 mouse monoclonal (1:1000), and caspase 9 mouse monoclonal (1:1000) (Initiator caspases sampler kit #12675), Drp1 rabbit monoclonal (#8570, 1:1000), GAPDH rabbit monoclonal (#2118, 1:5000), LC3B rabbit polyclonal (#2775, 1:1000), pan-actin rabbit polyclonal (#4968, 1:1000), PARP rabbit polyclonal (#9542, 1:1000), and PINK1 rabbit monoclonal (#6946, 1:500) antibodies were obtained from Cell Signaling Technology (Danvers, MA). ATG7 rabbit polyclonal antibody (PM039, 1:1000) was obtained from MBL International Corporation (Woburn, MA).

As previously described (24 (link), 25 (link)), proteins were extracted and normalized to at least 1500μg/ml. Proteins were subsequently analyzed on SDS-PAGE gels (8% or 10%, as appropriate) and transferred to polyvinylidene difluoride (PVDF) membranes for western blotting (24 (link), 25 (link)).
Primary antibodies that were utilized in this study are: SV40 LTAg mouse monoclonal (#554149, 1:1000, BD Biosciences), DNMT1 rabbit polyclonal (#5032, 1:1000, Cell Signaling Technology), HA-11 mouse monoclonal (#14921901, 1:500, Covance/Lab Corporation), CD71 mouse monoclonal (#sc-51829, 1:250, Santa Cruz Biotechnology), FTH1 rabbit polyclonal (#3998, 1:500, Cell Signaling Technology), pan-Actin rabbit polyclonal (#4968, 1:1000, Cell Signaling Technology), PARP rabbit polyclonal (#9542, 1:1000, Cell Signaling Technology), PAX-8 rabbit polyclonal (#10336-1-AP, 1:1000, Proteintech), Claudin-1 rabbit polyclonal (#187362, 1:10,000, Invitrogen), BAK rabbit monoclonal (#12105, 1: 1000, Cell Signaling Technology), Bcl-2 rabbit monoclonal (#2570, 1:1000, Cell Signaling Technology), Occludin-1 mouse monoclonal (#611090, 1:250, BD Biosciences), Cyclin D1 rabbit polyclonal (#sc-718, 1:1000, Santa Cruz Biotechnology), and Cyclin E mouse monoclonal (#sc-247, 1:500, Santa Cruz Biotechnology).

SDS-PAGE and western analyses were completed as previously described^{89 (link),90 (link)}. Briefly, normalized protein samples were loaded onto either 8% or 10% SDS-PAGE gels (as appropriate) and western blotting was completed with the following antibodies from Cell Signaling Technology (Danvers, MA, USA): (1) EVI1 rabbit monoclonal (#2593, 1:500), (2) Myc rabbit monoclonal (#13987, 1:500), (3) total Ras rabbit monoclonal (#3339, 1:1000), (4) β-catenin rabbit polyclonal (#9587, 1:1000), (5) BMI1 rabbit monoclonal (#6964, 1:1000), (6) LC3B rabbit polyclonal (#2775, 1:1000), and (7) Pan-Actin rabbit polyclonal (#4968, 1:500). SV40 LTAg mouse monoclonal (#554149, 1:1000) and p62 mouse monoclonal (#610832, 1:1000) antibodies were obtained from BD Biosciences (San Jose, CA, USA). FANCD2 mouse monoclonal (#sc-20022, 1:1000), Cyclin D1 rabbit polyclonal (#sc-718, 1:1000), and FoxJ1 mouse monoclonal (#sc-365216, 1:500) were obtained from Santa Cruz Biotechnology.