Gfp ab290

Tissues were fixed in 4% PFA overnight, paraffin processed or embedded into OCT for frozen sections, and sectioned. Hematoxylin and eosin staining was performed for routine histology. Antigen retrieval was performed using citrate or proteinase K for immunohistochemistry. The Tyramide Signal Amplification Kit (#NEL700A001KT; Perkin Elmer) was used according to the manufacturer instructions. Primary antibodies were incubated overnight and secondary antibodies were incubated for 1 h. The following antibodies were used: phosphohistone H3 (#9701, 1:100; Cell Signaling), CD8a (16-0081, 1:500; eBioscience), GFP (ab290, 1:500; Abcam), biotinylated antirat (#112-067-003, 1:300; Jackson), biotinylated antirabbit (#E0431, 1:300; Dako), and biotinylated antigoat (#305-067-003, 1:300; Jackson). Fluorescent antibodies 488-antirabbit, 568-antirat, and 647-antirat were from Molecular Probes (#A11008, #A11077 and A-21247, 1:600; Invitrogen), with fluorescent microscopy performed on a Keyence BZ-X700. Image analysis was performed using ImageJ.

AR (C-19, sc-815, Santa Cruz Biotechnology and clone G122-434, BD), PSA (A0562, Dako), IKBKE (D20G4, Cell Signalling), α-tubulin (clone DM1A, T9026, Sigma), LATS2 (kpm C-2, sc-515579 Santa Cruz Biotechnology), YAP (G-6, sc-376830 Santa Cruz Biotechnology), c-MYC (ab56, Abcam and N262, sc-764, Santa Cruz Biotechnology), TMPRSS2 (H-4, sc-515727, Santa Cruz Biotechnology), PARP1/2 (clone H250, sc-7150, Santa Cruz Biotechnology), FKBP5 (D-4, sc-271547, Santa Cruz Biotechnology), GFP (ab290, AbCam) Ki67 (clone MM1, Novocastra, Leica Biotechnology).

Cells were rinsed with ice-cold PBS and protein lysates were harvested in 1×lysis buffer (#9803, CST) supplemented with 1 mM PMSF. Lysates were first incubated with Pierce Protein A/G Magnetic beads (#88802, Thermo) at 4°C for 1 hr. Pre-cleared lysates were collected for IP input control and the rest were then incubated with Normal Rabbit IgG Polyclonal Antibody control (#12–370, Millipore) or primary antibodies against Cul3 (A301-109A, Bethyl Lab); Flag (M2 affinity gel, A2220, Sigma); GFP (ab290, Abcam); HA (clone C29F4, CST); Myc (clone 71D10, CST) at 4°C overnight. Antibody/lysates were further incubated with magnetic beads at RT for 30 min. The complex was washed with 1×lysis buffer for at least three times before added to elution buffer, which was prepared using 3×Blue Loading Buffer mixed with 30×DTT at 10:1 ratio (#7722, CST). Samples were boiled at 95°C for 5 min and supernatants were collected after centrifugation at 14,000 rpm at 4°C for 1 min. For western blot, mouse anti-rabbit Conformation Specific antibody (clone L27A9, CST) was used instead of traditional secondary antibodies.

All antibodies were used at a 1:1000 dilution in 5% nonfat milk for immunoblot. PE Mouse IgG1 (555749), PE Mouse Anti-Human CD13 (560998), PE Mouse Anti-Human CD133 (566593), and PE-CF594 Mouse Anti-Human CD326 (565399) were purchased from BD Biosciences. GASC1(sc-98678), c-Myc (sc-40), and SOX9 (sc-166505) were purchased from Santa Cruz Biotechnology. Nanog (ab109250), OCT4 (ab19857), SOX2 (ab97959), FBXO42 (ab81638), and GFP (ab290) were purchased from Abcam. Cleaved caspase-3 (9664), cleaved caspase-7 (8438), cleaved caspase-9 (7237), cleaved caspase-PARP (5625), Myc tag (71D10) (2278S), HA tag (2367), ROCK1 (4035), ROCK2 (9029), BCL2 (2872), ubiquitin (3936), Ki-67(9449), p-MLC2 (3671), GAPDH (2118), Rho-GTPase Antibody Sampler Kit (9968), Active Rho Detection Kit (8820), and NF-κB p65 Antibody Sampler Kit (4767) were purchased from Cell Signaling Technology.

Cell lysates were run on 5% or 8% polyacrylamide gels and subjected to Western blotting as described previously [6] (link). Antibodies used for Western blot, confocal immunofluorescence microscopy (IF) and immunoprecipitation were; GFP (ab290), GFP-Sepharose (ab69314) (Abcam), Vinculin (Sigma), Emerin (VP-E602) (Vector Labs), lamin A/C (sc-6215) (Santa Cruz), total β-catenin, active β-catenin clone 8E7 (05-665) (Millipore), nesprin-2 CH3 and nesprin-2 N3 (Immune Systems). N2CH3 peptide blocking experiments were performed as described previously using the peptide KRDLDELKDHLQL (Immune Systems) [6] (link). Filamentous actin was observed by IF using Rhodamine phalloidin (Invitrogen). Secondary antibodies for WB were horseradish peroxidase-conjugated anti mouse (NA931) or anti rabbit (NA94V) antibodies from GE Healthcare. ECL chemiluminescent kit (RPN2132, GE Healthcare) was used for detection according to manufacturer's instructions. Invitrogen anti-mouse Alexa fluor 568 (A11031) and anti-rabbit Alexa fluor 488 (A11034) were used as IF secondary antibodies. For IF cells were cultured on cover slips, fixed in 4% paraformaldehyde (Sigma), permeabilised in 0.5% NP-40 (Sigma) and processed as described previously [6] (link). All images were captured at 63 × magnifications using a Leica SP5 laser scanning confocal microscope.