Goat anti mouse hrp

Samples were separated via SDS-PAGE and transferred to nitrocellulose (pore size: 0.45 μm; Amersham Protran) or PVDF (pore size: 0.45 μm; Amersham Hybond) membranes. These blots were blocked for 1 h in 5% (w/v) skim milk-PBST (0.1% Tween in phosphate-buffered saline [PBS]), incubated for 1 h with appropriate primary antibodies (diluted in 5% skim milk-PBST) at room temperature, washed, and then incubated for 1 h with appropriate secondary antibodies (diluted in 5% skim milk-PBST) at room temperature. Chemiluminescence was detected with EZ-ECL reagents (Cyanagen). The optimal dilution for each antibody was determined as follows: mouse anti-DnaK (Abcam), diluted 1:1000; mouse anti-JNK (BD Pharmingen), diluted 1:1000; and mouse anti-actin (MPBio), diluted 1:10,000. Antibodies directed against T3SS components, including mouse anti-EspB, and mouse anti-Tir, were a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada) and Prof. Rebekah Devinney (University of Calgary, Canada). Horseradish peroxidase-conjugated (HRP)-goat anti-mouse (Abcam), diluted 1:10,000, was used as the secondary antibody for these analyses. Blots representative of at least three independent experiments are presented in the results section.

The total protein concentration of MACS-isolated EVs was quantified using Lowry Protein Assay [28 (link)]. 10 µg of the EV fractions were lysed directly by sodium dodecyl sulfate (SDS) sample buffer (200 mM Tris-HCL (pH 6.8); 10% SDS; 0.4% bromophenol blue; 40% glycerol) and was separated by SDS (12%) gel electrophoresis (SDS-PAGE). For the detection of CD9 and TSG101 signal the EV fraction proteins were transferred onto 0.45 µm PVDF-membranes (Cytiva, 10600023, Marlborough, MA, USA). Membranes were blocked with 5% milk powder and 0.1% Tween in Tris-Buffered Saline (TBS) for 1 h at room, followed by primary antibody incubation overnight at 4 °C. The horseradish peroxidase (HRP)-coupled secondary antibodies were incubated for 1 h at room temperature. Subsequently, enhanced chemiluminescence (Millipore WBKLS0500, Merck KGaA, Burlington, MA, USA) detection and visualization were implemented and detected by Octoplus QPLEX imager from NH DyeAgnostics (Halle, Germany). For semi-quantitative analysis of western blot signals, ImageJ software was used. The following antibodies were applied: CD9 (1:1000 Purified mouse monoclonal anti-human, Mouse IgG1, κ, 312102 Biolegend, San Diego, CA, USA), TSG101 (1:200 tsg 101 mouse monoclonal antibody, sc-136111, Santa Cruz, Dallas, USA), goat-anti-mouse-HRP (1:1000 dilution, polyclonal, ab205719, Abcam, Cambridge, UK).

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).

Cell pellets from patient and control fibroblasts were lysed using RIPA buffer and Protein Inhibitor Cocktail on ice, and protein levels were measured using a Bradford Assay. Twenty micrograms of protein was run on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. Membranes were incubated overnight at 4 °C, with mouse anti-Drp1 (Abcam; 1:1000), mouse anti-OPA1 (BD Biosciences; 1:1000), mouse anti-Mfn1 (Abcam; 1:1000) or rabbit anti-Mfn2 (St Johns Laboratory; 1:500). Membranes were also probed for β actin as a loading control (St Johns Laboratory; 1:1000). Membranes were then incubated with goat anti-mouse HRP (Abcam; 1:10,000) or goat anti-rabbit HRP (Dako; 1:5000), as appropriate. Membranes were imaged using the G box chemi system using GeneSnap software (Syngene). Densitometry was analyzed using GeneTools software (Syngene).

Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [77 (link)], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used. Ninety micrograms of extracts were loaded to analyze the specific proteins from zebrafish embryos. The signal intensity of the band was analyzed using ImageJ software.
To reduce biased data, we only took into account the intensity of ANP32a when the expressional intensity of the internal control, alpha-tubulin, loaded into each well on gel was almost identical on the same blot paper.

The protein samples were prepared from the cell pellets as previously described [18 (link),19 (link)]. Then the samples were separated on SDS-PAGE gel and transferred to NC (nitrocellulose, NC) membrane. After blocking in 5% nonfat milk, primary antibodies were added and incubated with membrane overnight at 4 °C (anti-Flag (1:5000, M2, Sigma), anti-DNMT3B (1:1000), anti-actin (1:10,000, clone C4, Sigma)). Secondary antibody was incubated for 1 h at room temperature (goat anti-mouse HRP (1:5000, Abcam), goat anti-rabbit (1:10,000, Abcam)). The images of Western blot were captured by image lab version 5.1 (Bio-Rad Laboratories, Hercules, California, USA).

Tumours harvested for paraffin embedding were fixed for 2 h in 4% paraformaldehyde (PFA) or 10% formalin and then embedded in paraffin using standard protocols. 4 µm sections underwent antigen retrieval and were stained using the DAKO Autostainer (K8012) as described previously^{14 (link)}. Sections were incubated for 30 min with Ki67 antibody (1:200) (DAKO M7240) in 5% BSA in Tris Buffered Saline followed by goat anti-mouse HRP (Abcam)(1:250) and staining with 3,3′-diaminobenzidine DAB (1 drop per ml). Haematoxylin staining was performed on all the slides to assist in distinguishing between tumour and chick nuclei. A total of 9 fields from 3 slides were counted per tumour and at least three tumours per condition were analysed.