Cleaved caspase 3 rabbit mab

cDNA was synthesised using High‐Capacity RNA‐to‐cDNA™ Kit (Applied Biosystems #4387406). qRT–PCR was performed using Taqman gene expression assay (Thermo Fisher #4304437) with following Taqman primers (Thermo Fisher): Hs01045540_g1 (ARC), Hs00156548_m1 (CLU), Hs00610256_g1 (DUSP1), Hs01044001_m1 (DUSP6), Hs00152928_m1 (EGR1), Hs00166165_m1 (EGR2), Hs00170630_m1 (FOS), Hs00171851_m1 (FOSB), Hs04187685_m1 (FOSL1), Hs00357891_s1 (JUNB), Hs00374226_m1 (NR4A1), Hs00943178_g1 (PGK1), Hs00169585_m1 (PPP1R15A), Hs00153133_m1 (PTGS2), Hs04334126_m1 (TFPI2), Hs00959047_g1 (TNFRSF12A), Hs00381614_m1 (ZCCHC12), Hs00185658_m1 (ZFP36).
Protein was extracted using Bio‐Rad Cell Lysis Buffer (#171‐304006M). Concentration was determined using Thermo Fisher Pierce BCA Protein Assay (#23228). 25–50 μg of purified protein was used for blotting. Images were acquired using Li‐Cor Odyssey Scanner. Western blot antibodies were as follows: EGR1 (Santa Cruz sc‐110), FOS (Cell Signaling #2250), CLU (Santa Cruz sc‐8354), FOSL1 (Santa Cruz sc‐376148).
For flow cytometry, cells were harvested 48 h after treatment and fixed in 2% paraformaldehyde (PFA) for 10 min at RT. Cells were permeabilised in methanol and incubated on ice for 30 min. For immunostaining, cells were incubated for 1 h with Cleaved Caspase‐3 rabbit mAb (Cell Signaling #9602).

Protein lysates from tissue samples or culture cells were extracted by RIPA lysis buffer (Beyotime, China) with protease inhibitors (Sigma, USA). The protein concentration was determined by BCA kit (Sigma, USA). A total of 30 μg proteins were loaded to each well and separated by 10% or 15% SDS-PAGE. Next, proteins were transferred to nitrogen membranes (Thermo Fisher Scientific, USA) and blocked by 5% non-fat milk for 1 h at room temperature. The membranes were stained with specific first antibodies at 4 ˚C overnight and corresponding second antibodies for 1 h at room temperature. The antibodies were: Anti-SFRP2 rabbit IgG (Abcam#ab137560, 1: 1000), Cleaved Caspase-3 Rabbit mAb (Cell signaling#9664, 1: 1000), GAPDH Rabbit mAb (Cell signaling#5174 1: 1000), β-Catenin Rabbit mAb (Cell signaling#8480, 1: 1000), Active β-Catenin Rabbit mAb (Cell signaling#19807, 1: 1000), GSK-3β Rabbit mAb (Cell signaling#12456, 1: 1000), Phospho-GSK-3β (Ser9) Rabbit mAb (Cell signaling#5558, 1: 1000) and goat anti-rabbit IgG-HRP (Cell signaling #7074; 1:4000).

Immunofluorescence observations were performed to investigate cleaved caspase-3 expression in the LEO treated cell. After treatments with LEO and puromycin at EC₅₀ concentrations for 24 h, cells were processed, as reported in the literature [45 (link)], and subsequently incubated with a cleaved caspase-3 rabbit mAb (Cell Signaling Technology, Leiden, The Netherlands) diluted to 1:400 in a blocking buffer (5% BSA in PBS) for 1 h at room temperature. After rinsing three times in PBS, the coverslips were dried and incubated with an Alexa Fluor^® 488 Goat Anti-Mouse IgG Antibody (Molecular Probes, Eugene, OR, USA) diluted to 1:100 in a blocking buffer. For evaluation of possible unspecific staining, control samples were prepared by omitting the primary antibody. After three washings in PBS, nucleic acid staining was obtained by incubation with Fluoroshield with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Merck KGaA, Darmstadt, Germany), and then samples were placed. The images were captured by a Zeiss LSM 710 Confocal Microscope (Carl Zeiss, Oberkochen, Germany.

Total proteins were extracted using Complete Lysis buffer containing, proteases inhibitors (04719956001 Roche) and phosphatase inhibitors (04906845001 Roche). Preparation of nuclear extracts was performed by using NE-PER™ nuclear and cytoplasmic extraction reagents according to the manufacturer’s instructions. The Western blot of cell lysates was performed as previously described [40 (link)]. Antibodies: p-YAP rabbit mAb (#13008), YAP rabbit polyclonal (#4912), P-FAK rabbit (#2383), Cleaved caspase-3 rabbit mAb (#9664), cleaved PARP rabbit polyclonal (#9541), and P-AKT rabbit mAb (#4060) all purchased by Cell Signalling (MA, USA); and P-ERK mouse mAb (#sc-7383, Santa Cruz Biotechnology, USA), HDAC1 rabbit Ab (#H3284, Sigma-Aldrich), anti-β-tubulin (HRP) rabbit polyclonal (ab21058) and anti-GAPDH rabbit polyclonal antibodies (ab9385) from Abcam (Cambridge, MA, USA). Antibody binding was revealed by ECL Prime (RPN2232, GE Healthcare, Milan, Italy) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK).