Goat anti rabbit hrp

Equal amounts of proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted, and membranes were first probed with anti-VEGF antibody (rabbit polyclonal 1:2000, Abcam) and reprobed with β-actin antibody (mouse monoclonal 1:1000, Santa Cruz). Goat anti-mouse HRP and goat anti-rabbit HRP (BioRad) were used as secondary antibodies. Western blots were revealed by enhanced chemiluminescence (Amersham). Scanned bands were quantified using ImageJ software Version 1.43 (National Institutes of Health, http://rsb.info.nih.gov/ij/). All Western blot results were evaluated by densitometric scanning, corrected with respect to β-actin expression, and expressed relative to the value obtained with the corresponding control (arbitrarily set as 1). Equal protein loading was assessed by anti–β-actin immunoblotting.

Cytoplasmic and nuclear protein were extracted from 5 × 10⁶ OCI-AML3 cells exposed to OTX015 (MK-8628), JQ1, ATO + ATRA or 0.1% DMSO with NE-PER Nuclear and Cytoplasmic kit (#78833, Life Technologies, Courtaboeuf, France). Proteins thus obtained were concentrated with Amicon Ultra centrifugal filters, Ultracel 30 K and 10 K (#UFC503096 and #UFC501096, Merck Millipore, St. Quentin Fallavier, France). Thirty microgrammes of cytoplasmic proteins and 5–10 µg of nuclear proteins were loaded on SDS-polyacrylamide gels using 4–15% gradient gels (Bio-Rad, Marnes-La-Coquette, France) and transferred to nitrocellulose membranes using a Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad, Marnes-La-Coquette, France). Membranes were blocked with LI-COR blocking buffer and incubated with the primary antibody overnight at 4 °C: anti-NPM1m (#ab65816, Abcam, Paris, France), anti-NPM1total (#H00004869-M01, CliniSciences, Nanterre, France), anti-BRD4 (#ab128874, Abcam, Paris, France), anti-tubulin (#ab176560, Abcam, Paris, France), or anti-lamin (#ab16048, Abcam, Paris, France). Secondary antibodies were goat anti rabbit HRP or goat anti mouse HRP (Bio-Rad, Marnes-La-Coquette, France), incubated for 1 h at room temperature. Bands were visualized using the ChemiDoc Touch (Bio-Rad, Marnes-La-Coquette, France).

Frozen colon tissue (~100 mg) was homogenized in cell lysis buffer (500 μl) (Cat EPX-99999-000 eBioscience, San Diego, USA) containing 0.1% proteinase inhibitor cocktail (Sigma, St. Louis, MO) and 1% NP40, in ceramic bead tubes using Fast Prep-24^TM 5G machine (M.P. Biomedical LLC, California, USA) at a speed 6.0 ms for 30 s (twice). Samples were centrifuged at 12,000 g for 15 min at 4 °C, supernatants were collected and protein concentration was determined using Bio-Rad protein estimation kit (BioRad). 100 μg of protein was subjected to Western Blot analyses using 8% acrylamide gel. Membranes were probed with anti-pig primary antibodies raised in rabbit overnight at 4 °C and subsequently incubated with 1:10,000 dilution of goat-anti-rabbit HRP for 1 h at room temperature (BioRad Laboratories Inc., California). We used 1:1000 dilution of anti-VE-cadherin, anti-catenin, anti-HSP 27 (Abcam, Cambridge, MA) as primary antibodies. Detection was performed using a chemiluminescence system (super signal west chemiluminescent substrate, Thermo Scientific). Image Quant software (Image Quant TL 8.1 Version) was used for densitometric analysis. Anti-rabbit vinculin (Abcam ab73412) that cross reacts with pigs was used as a housekeeping protein for normalization of a target protein.

The following antibodies were purchased from the indicated vendors: rabbit monoclonal antibodies against β-actin (ABclonal, AC038); mouse monoclonal antibodies against SNAP-25 (Synaptic systems, Cl 71.1) or Syt-1 (Synaptic systems, #105011); rabbit polyclonal antibody against SV2C (Synaptic systems, #119202). SV2 mouse monoclonal antibody (pan-SV2) was generously provided by E. Chapman (Madison, WI) and is available from Developmental Studies Hybridoma Bank (AB_2315387). Secondary antibodies were purchased from the following vendors: goat anti-rabbit-HRP (Bio-Rad, 1705046) and goat anti-mouse-HRP (Abcam, ab97023).
BoNT/E utilized for cell-based assays was purchased from Metabiologics or List Biologics (#141A) by the Dong lab. No recombinant BoNT/E was imported into the United States. All active BoNTs are stored in a locked freezer. Used toxins and contaminated media/reagents/containers are exposed to a 10% bleach solution for decontamination. Lentiviral constructs (in Lox-Syn-Syn vector) encoding full-length rat SV2A and SV2C were previously described^{25 (link)}. Rat SV2Ac chimera was generated by replacing F487–E532 of SV2A with V473–K518 of SV2C using Gibson assembly and subcloned into Lox-Syn-Syn vector. SV2A (Y535T/Y557E) and SV2C (T521Y/E543Y) were generated by site-directed mutagenesis through overlapping PCR. All constructs were confirmed by sequencing (Genewiz).

Cells were lysed on ice with lysis buffer containing 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and protease inhibitors (Millipore). 0.5 × 10⁶ cells were lysed for each condition and run on SDS-PAGE gels, transferred to nitrocellulose (Bio-Rad), and blotted using standard procedures. Rat anti-mouse Bim mAb (3C5) was purchased from Enzo Life Sciences. Goat anti-rat HRP was purchased from BioLegend. Rabbit anti-mouse actin mAb (D18C11) was purchased from Cell Signaling. Goat anti-rabbit HRP was purchased from Bio-Rad.

Western blots were performed as described previously^{82 (link),169 (link)}. The following antibodies were used:
Rabbit anti-UHRF1 (polyclonal; 1:250; Cell Signalling, D6G8E), mouse anti-alpha-Tubulin (monoclonal; 1:500; Sigma, T9026), rabbit anti-H3 (polyclonal; 1:1000; Abcam, ab1791), mouse anti-GFP (monoclonal; 1:1000; Roche), mouse anti-FLAG M2 (monoclonal; 1:1000; Sigma, F3165), rabbit anti-xDNMT1 (polyclonal;^{82 (link)}), rabbit anti-xUHRF1 (polyclonal;^{82 (link)}), rabbit anti-USP7 (polyclonal; Bethyl Lab., A300-033A), rabbit anti-H3 (polyclonal; Abcam, ab1791), rat anti-TET1 (monoclonal; 1:10;^{176 (link)}), rat anti-alpha-Tubulin (monoclonal; 1:250; Abcam, ab6160). goat anti-rat HRP (polyclonal; 1:1000; Jackson ImmunoResearch), goat anti-rabbit HRP (polyclonal; 1:1000; BioRad), mouse anti-xCDC45 (monoclonal;¹⁷⁷ ), mouse anti-xRPA2 (monoclonal;^{178 (link)}), and mouse anti-PCNA (monoclonal; Santa Cruz, sc56). Uncropped and unprocessed scans of blots can be found in the Source Data file.