Horseradish peroxidase hrp conjugated anti mouse igg

NIH3T3 cells (CRL-1658; ATCC, Manassas, USA) were transiently transfected with 1 μg of 2PLEASE plasmid and 1 μg of either pBS185CMV-Cre (ADDGENE #11916) or an empty vector (a negative control), using FuGENE HD Transfection Reagent (Promega, Madison, USA), according to the manufacturer's instructions. Cells were harvested at 2 days post-transfection and lysed in 1 × RIPA buffer (#9806; Cell Signaling). Western blotting experiments were performed using standard methods. Anti-p53 (#2524), anti-GFP (#2555) and anti-LacZ (#2372) primary antibodies were purchased from Cell Signaling Technology, and anti-Kras (sc-30) and anti-β-actin (sc-47778) were purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (sc-2005, Santa Cruz) and anti-rabbit IgG (A0545, Sigma) were used as secondary antibodies. Fluorescence imaging of transfected cells was performed at 2 days post-transfection, using an inverted microscope (IX71, Olympus, Japan). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions.

We obtained rSjP40 protein as previously described (Chen et al., 2016b (link)). Mouse mAbs against COL1A1 (Abcam, United Kingdom), rabbit mAbs against GAPDH (Goodhere, China) or Ets-1 (Proteintech, United Kingdom), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Santa Cruz, United States) and HRP-conjugated anti-rabbit IgG (Biosharp, China) were purchased from the indicated companies. Plasmids containing COL1A1 promoter sequences constructed as previously described (Chen et al., 2019a (link)) were preserved in our lab.

Vps35 mutant mice have been described previously [13 (link), 21 (link), 25 (link)]. Mice were maintained on a standard rodent diet and in a standard facility at Augusta University. Animal care was approved by the Institute of Animal Care and Use Committee (IACUC) at the Augusta University according to the National Institute of Health guidelines.
The following reagents were used: mouse anti-Neuronal Class III ß-Tubulin (Thermo Fisher Scientific, Catalog #:32–2600); rabbit anti-Ki67 antibodies (Abcam, Catalog #: ab15580); chicken anti- ß-galactosidase antibody antibodies (Abcam, Catalog #: ab9361) rabbit anti-SLC4A11 antibodies (Thermo Fisher Scientific, Catalog #: PA5-53730). Rabbit polyclonal anti-Vps35 antibody was generated against the murine Vps35 C-terminal sequence by Cocalico Biologicals, Inc. (PA, USA) as described previously [13 (link)]. Phalloidin-fluorescent conjugates were purchased from Molecular Probes (Thermo Fisher Scientific, Catalog #:A12379). For immunofluorescence analysis, the secondary antibodies were Alexa Fluor-488 or Alexa Fluro-594 conjugated anti-mouse or anti-rabbit antibodies (Invitrogen). For Western analysis, the secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Santa Cruz Biotechnology, Inc.). All cell culture reagents were purchased from Thermo fisher.

CLPr was prepared from cacao liquor and its composition was previously described.^{(17 (link),19 (link))} Glucose was measured using a commercially available kit [Labassay Glucose Wako kit (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan)]. Antibodies against β-actin, AMPKα, phospho-AMPKα (thr172), phospho-CaMKK2 (ser511), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), liver kinase B1 (LKB1) and phospho-LKB1 (ser428) were purchased from Cell Signaling Technology Co. (Denver, MA). Antibodies against CaMKK2 and GLUT4 were purchased from Abcam (Hercules, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG, HRP-conjugated anti-goat IgG antibodies, anti-insulin receptor (IR), and anti-Lamin B were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated anti-rabbit IgG was from Bio-Rad Laboratories Inc. (Hercules, CA). All other reagents used were of the highest grade available from commercial sources.

The following antibodies were used: anti-G6PD (Genesis Biotech Inc., New Taipei, Taiwan), anti-E-cadherin, anti-N-cadherin, anti-β-catenin (BD Biosciences, San Jose, CA, USA), anti-β-actin (GeneTex Inc., Irvine, CA, USA), alexa fluor 488 goat anti-mouse IgG, alexa fluor 594 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), anti-ZEB1, anti-Smad3 (Cell Signaling Technology, Danvers, MA, USA), anti-p22_phox (Abcam, Cambridge, UK), horseradish peroxidase (HRP)-conjugated anti-mouse IgG, and anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Recombinant human TGF-β1 was purchased from PeproTech (Rocky Hill, NJ, USA). The g6pd targeting and nontarget Morpholinos were purchased from Gene Tools (Philomath, OR, USA).

5-Aza-2′-deoxycytidine (5-aza-dC) and 5-Fluorouracil (5-FU) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Cell Count Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Tokyo, Japan). The TRIzol was purchased from Invitrogen. The ECL western blotting kit was obtained from Amersham (Arlington Heights, IL, USA), and Immobilon-P membranes were obtained from Millipore Corp. (Bedford, MA, USA). Anti-p53 and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the anti-CADM1 antibody was obtained from Abnova (Walnut, CA, USA), the anti-phospho-p53 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), and horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were obtained from Santa Cruz Biotechnology.

RY10-4 (Figure S1) was synthesized previously in our laboratory [6 (link)], and the structure was confirmed by NMR and MASS. Purity (95%) was measured by HPLC analysis. RY10-4 was dissolved in dimethyl sulfoxide (DMSO) to make a 10 μM stock solution, and this was stored at −20°C. The working dosage was freshly prepared in basal medium with a final DMSO concentration of less than 0.1%. The antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the antibodies against AKT, p-AKT, Notch1, NICD, and HES1 were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against Dll4 was purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA).