Hrp conjugated anti mouse igg

Mouse monoclonal anti-TFRC antibody (Zymed, 136800), rabbit polyclonal cleaved CASP3 (Asp-175) antibody (Cell Signaling Technology, 9664), rabbit polyclonal anti-ATG5 antibody (Cell Signaling Technology, 8540), rabbit monoclonal anti-phospho-TBK/NAK (Ser172) antibody (Cell Signaling Technology, 5483), rabbit monoclonal anti-TBK1/NAK antibody (Cell Signaling Technology, 3504 and Abcam, ab40676), mouse monoclonal anti-HA antibody (Roche Applied Biosystems, 11583816001), rabbit polyclonal anti-HA (Santa Cruz, sc-805), rabbit polyclonal and mouse monoclonal anti-GFP (Santa Cruz, sc-9996, sc-8334), anti-GAPDH (Millipore, MAB374) and anti-actin (Millipore, MAB1501) are commercially available. Secondary antibodies used were Cy-3-conjugated anti-mouse IgG (Amersham, PA43002), Cy-3-conjugated anti-rabbit IgG (Amersham, PA43004), HRP conjugated anti-mouse IgG (Amersham, NA9310), HRP conjugated anti-rabbit IgG (Amersham, NA934), Alexa-488 anti-mouse and anit-rabbit IgG (Molecular Probes, A21202, A21206). Phospho-OPTN antibody specific to S177 residue of OPTN was a generous gift from Dr. Ivan Dikic of Goethe University Medical School, Theodor-Stern-Kai 7 60590 Frankfurt am Main / Germany [27 (link)]. BX-795 (Calbiochem, 204001) and chloroquine (Sigma, C6628) are commercially available.

RWPE1 cells were seeded (4 × 10⁵ cells/well) into 6-well plates (BD Falcon, USA) and exposed to 100 nM of DHT and TBECH-γδ alone or in combination with 20.0 µM of either hydroxyflutamide or DPTE for 6 days. Cells were lysed in RIPA buffer and protein was extracted. Following quantification, the proteins were transferred onto PVDF membranes (Amersham Biosciences, UK) as described previously^{18 (link)}. The membranes were rinsed for 30 min with Tris-buffered saline-Tween (0.1%) and incubated overnight at 4 °C with mouse anti-PSA antibody (Sigma, USA) at 1:500 dilutions. The secondary antibody, HRP-conjugated anti-mouse IgG (Amersham Biosciences, UK) was incubated for 1 hr at 1:5000 dilution at RT. Detection was performed using the enhanced chemiluminescent method (Amersham Biosciences, UK). The membrane was then stripped and probed for β-actin using mouse anti β-actin antibody (Sigma, USA). The bands were analyzed and quantified using ImageJ software (National Institute of Health, USA) and normalized with their respective β-actin level.

Reagent sources were as follows: MCT (Wako Pure Chemical Industries, Ltd., Osaka, Japan), l-NAME (Dojindo, Kumamoto, Japan), BAY11-7082 (Merck (Calbiochem), Darmstadt, Germany), PD98059 (Wako Pure Chemical Industries, Ltd.), SP600125 (Jena Bioscience, Jena, Germany) and SNP (Sigma Aldrich, St., Louis, MO, USA).
Antibody sources were as follows: anti-POSTN (1:100 dilution) (Proteintech, Rosemont, IL, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (1:1000 dilution) (GeneTex, Irvine, CA, USA), anti-iNOS (1:250 dilution for western blotting or 1:100 dilution for immunohistochemistry) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA or Bioss, Woburn, MA, USA), anti-total-actin (1:1000 dilution) (Sigma Aldrich), anti-phospho-VASP (1:500 dilution) (Abcam, Cambridge, UK), anti-phospho-ERK1/2 (1:1000 dilution), anti-phospho-JNK (1:250 dilution), anti-total-JNK (1:500 dilution), anti-phospho-NF-κB p65 (1:500 dilution) (Cell Signaling Technology, Madison, WI, USA), anti-total-ERK1/2 (1:100 or 1:200 dilution) (Santa Cruz Biotech, Santa Cruz, CA, USA or Bioss), anti-total-NF-κB p65 (1:500 dilution) (Santa Cruz Biotech), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (1:10,000 dilution) (Amersham Biosciences, Buckinghamshire, UK or Cell Signaling Technology).