Anti mouse igg hrp

Total protein extracts were separated in 5%–20% gradient acrylamide gel for SDS-PAGE, transferred to a nitrocellulose membrane and probed with specific antibodies against phospho-Akt (pAkt; phospho-Ser473, Cell Signaling, 1:1,000, Cat no. 4051, raised against synthetic phosphopeptide corresponding to residues around Ser473 of mouse Akt), Akt (40D4, Cell Signaling, 1:2,000, Cat no. 2920, detects the C-terminus of the protein), InsR (4B8, Cell Signaling, 1:1,000, Cat no. 3025, raised against synthetic peptide corresponding to residues surrounding Tyr999 of human insulin receptor β), IGF-1R (Cell Signaling, 1:1,000, Cat no. 3027, detects the C-terminus of the protein) and mouse anti-β-actin (Sigma-Aldrich Cat no. A5441), at 4°C for 12 h. The membranes were initially blocked with 5% nonfat milk or BSA in TBS-T (0.25 M Tris-Base, 8% NaCl, pH 7.6, 0.1% Tween 20). The membranes were washed four times for 10 min in TBS-T and incubated with secondary anti-mouse IgG-HRP (Dianova, 1:20,000, Cat no.111-035-146) or anti-rabbit IgG-HRP (Dianova, 1:20,000, Cat no. 111-035-144) antibodies for 1 h 30 min. The membranes were washed again and developed with an ECL system (Luminata Western HRP Substrate from Millipore or SuperSignal West Pico from Thermo Fisher Scientific, Waltham, MA, USA).

Full-length Xl_Kif18A was cloned from egg cDNA using primers 5′-attaggccggcccatggaagcctcccaagaggacgtc-3′ and 5′-taatggcgcgccctcagggatctttaagggctgc-3′. Rabbit polyclonal antibodies were generated against a C-terminal peptide: N-CGGRKKAALKDP-C (Ab^18Apep), a protein fragment containing an N-terminal Serine: aa 845-953 (Ab^18A-C) and a fragment from aa 1-104 tagged with an N-terminal His6-SMT3 (Ab^18AN). Other antibodies used were: human Kif18A^N (Mayr et al., 2007 (link); 1 µg/ml), anti-GFP (Covance, MMS-118P, 1:1000); anti-cyclin B2 (Abcam, ab18250; 1:1000), anti-XErp1 (Schmidt et al., 2005 (link); 1 µg/ml); anti- c-Mos (Santa Cruz, C237; 1:250), anti pY15-Cdk1 (Cell Signaling, 9111, 1:1000); anti-Flag (Sigma, F1804, 1:1000); anti α-tubulin (Sigma, F2168, 1:2000), anti HURP (Abcam, ab70744, 1:500), anti-mouse IgG-HRP and anti-rabbit IgG-HRP (Dianova, 115-035-062 and 111-035-003, both 1:3000).

Proteins were transferred onto a nitrocellulose membrane and blocked in 5% milk in PBST for 30 min at 22°C. Membranes were reacted for 2–4 h at 22°C or overnight at 4°C with primary antibodies diluted in PBST. Primary antibodies used were as follows: rabbit anti-acSMC3 (1:1,000, MBL), rabbit anti-SMC3 (1:1,000, A300-060A; Bethyl Laboratories), mouse anti-SMC1β (1:2, hybridoma supernatant), guinea pig antimouse ESCO2 (1:500, [Whelan et al, 2011 (link)]), and mouse anti-GAPDH (1:200, sc-32233; Santa Cruz). Membranes were washed three times in PBST, and HRP-conjugated secondary antibodies were added for 1 h at 22°C in PBST. Secondary antibodies were all diluted 1:5,000 and were used as follows: antirabbit IgG HRP (18-8816-31; eBioscience), antimouse IgG HRP (115-035-003; Dianova), and anti–guinea pig IgG HRP (106-035-003; Dianova). A pre-stained protein ladder (#26619; Thermo Fisher Scientific) was loaded onto each gel to determine the molecular weight of the detected bands. Blots were washed three times in PBST and developed using chemiluminescent HRP substrate (WBKLS0100; Millipore) and imaged on a Kodak ImageStation 2000MM.