Sc 166545

SDS-PAGE and immunoblotting were performed as previously described [23 (link)]. Briefly 15 mg of retina were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking the membranes were incubated with the following primary antibodies: mouse anti-HSP25/27 (1/5000; SMC-163C/D, StressMarq), and anti-HSP70 (1/5000; SMC-114C/D, StressMarq), rabbit anti-LC3B (1/1000; #2775, Cell Signaling, MA 01915) and anti-Atg12 (1/1000; #2011, Cell Signaling). Mouse anti-GAPDH (1/500; SC-166545, Santa Cruz) was used as an internal control. Immunoblots were washed in TBS–0.02% Tween 20 (TBST), incubated for 1 h with the appropriate horseradish peroxidase-coupled IgG (diluted 1:5000), washed in TBS–0.02% Tween 20, revealed using Super Signal (Pierce, Rockford, IL), and exposed to Hyperfilm ECL (Amersham Biosciences). Silver staining using the Biorad Silver Stain Plus kit was performed according to the manufacturer’s instructions.

The following reagents used in this study were purchased from the indicated sources: cycloheximide (Sigma, 20 μg/ml), doxorubicin (Sigma, 0.5 μg/ml), doxycycline (Sigma, 1 μg/ml), Hoechst 33342 (Sigma, 1 μg/ml), lipofectamine 2000 (Invitrogen), streptavidin-coated agarose beads (Thermo Fisher Scientific), complete EDTA-free protease inhibitor cocktail (Roche Applied Science), antibodies against GAPDH (Santa Cruz, sc-166545, 1:5000), p53 (Santa Cruz, sc-126, 1:1000), p21 (Sigma, #P1484, 1:2000), Flag (Sigma, #F3165, 1:4000), p27 (Abcam, Y236, 1:1000), cyclin D1 (Santa Cruz, sc-753, 1:1000), cyclin E1 (Santa Cruz, sc-481, 1:1000), PTBP1 (Proteintech, 12582-1-AP, 1:1000), HRP-conjugated secondary antibodies against mouse (115-035-062), and rabbit (111-035-144) (Jackson ImmunoResearch, 1:10000).

The cell lines (MDA-MB-231, MDA-MB-468, SKBR3, BT-474) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human β-arrestin-1 siRNA (sc-29741), β-arrestin-2 siRNA (sc-29208), and scrambled oligo-ribonucleotide complex utilized as control (sc-37007), siRNA transfection reagent (sc-29528), and transfection medium (sc-36868) were all obtained from Santa Cruz Biotechnology. Human β-arrestin-1 (ARRB1) cDNA (SC303424), human β-arrestin-2 (ARRB2) cDNA (SC108950), TurboFectin 8.0 transfection reagent (TF81001), cloning vector PCMV6-XL6 (PCMV6XL6), and cloning vector PCMV6-XL5 (PCMV6XL5) were all purchased from OriGene Technologies, MD, USA. The antibodies, β-Arrestin-1 (sc-9182), β-Arrestin-2 (sc-13140), CDC45 (sc-55569, CCNB1 (sc-245), CCNB2 (sc-28303), BUB1 (sc-365685), GAPDH (1:5,000; Sc-166545), goat anti-rabbit (1:10,000; Sc-31460), rabbit anti-goat (1:10,000; Sc-2768), and donkey anti-mouse (1:10,000; Sc-2314) were obtained from Santa Cruz Biotechnology, CA, USA. β-Arrestin-1 (D803J), β-Arrestin-2 (C16D9), IGF-1R (1:1000; D23H3), E-Cadherin (1:1000; 24E10), Snail (1:1000; C15D3), and Vimentin (1:1000; D21H3) were obtained from Cell Signaling Technology, the Netherlands.