Mouse anti actin ab3280

Guide RNAs against ATP2C1 were designed and ordered as a single-stranded oligonucleotide from IDT (5′-GGAGCTGTCACCTTAGAACA-3′); the scrambled control guide (Scr; 5′-GGTCTCTGTACGGGCCGCCC-3′) does not align with the human genome by primer blast. Following phosphorylation and annealing, guides were ligated into BsmBI-digested LentiCrisprV2 (a gift from the Feng Zhang lab; Addgene plasmid number 52961). Mouse anti-actin (ab3280) was obtained from Abcam (Cambridge, MA), while goat-anti-mouse horseradish peroxidase (HRP) (32430) was obtained from Thermo Fisher. Rabbit anti-lamin B1 (12586) was obtained from Cell Signaling Technology (Danvers, MA). Hybridoma AAV antibodies A1, A69, and B1 were purchased from American Research Products, Inc. (Waltham, MA). Fluo4-am (no. F14201) was purchased from Thermo Fisher. Ionomycin (2092) and BAPTA-AM (2787) were obtained from Tocris Bioscience (Minneapolis, MN). Bortezomib (S1013) was obtained from Selleck Chemicals (Houston, TX).

Mouse anti-actin (ab3280) and mouse anti-KIAA0319L(AAVR) (ab105385) were obtained from Abcam (Cambridge, MA). Goat anti-rabbit-HRP (111-035-003) was obtained from Jackon ImmunoResearch (West Grove, PA). Anti-Ubiquitin antibody P4D1 (sc-8017) was purchased from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-RNF121 (PA5-61136), Goat anti-Biotin (31852), and Goat anti-mouse-HRP (32430) were obtained from ThermoFisher. Anti-capsid protein antibody B1 [54 (link)] was used to blot for capsid protein, while anti-capsid antibody A20 [55 (link)] was used for immunofluorescence. MG132 (10012628) was purchased from Cayman Chemical (Ann Arbor, MI). PYR-41 (N2915), DBeQ (SML0031), as well as ATM/ATR (118501) and DNAPK (260960) inhibitors were purchased from Sigma-Aldrich. PladB was a kind gift from the lab of Yasuhiro Ikeda (Mayo Clinic, Rochester, Minnesota).

Preparation of lysates and immunoblot analyses were performed as described previously (5 (link)) using Tris lysis buffer (50 mM Tris–HCl pH 7.8, 150 mM NaCl, 1% IGEPAL CA-630) containing 20 mM NaF, 20 mM β-glycerophosphate, 0.3 mM Na-vanadate, 20 μg/ml RNase A, 20 μg/ml DNase and 1/300 protease inhibitor cocktail (P8340, Sigma–Aldrich) and phosphatase inhibitor cocktail #2 (P5726, Sigma–Aldrich). Antibodies used in this study were purchased from the following sources: rabbit anti-Cdk2 (M2 SC-163, Santa Cruz Biotechnology), mouse anti-actin (ab-3280, Abcam, Cambridge, MA, USA), rabbit anti-GFP (ab-290, Abcam), mouse anti-tubulin (Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-Cyclin A (SC-751, Santa Cruz Biotechnology) and mouse anti-cyclin E (SC-198, Santa Cruz Biotechnology). Secondary antibodies used for western blot analysis were goat anti-mouse (31430) and, goat anti-rabbit (31460, Thermo Scientific). mouse anti-tubulin hybridoma cell line (clone #12G10) was developed by J. Frankel and E.M. Nelson under the auspices of the NICHD and maintained by the Developmental Studies Hybridoma Bank.