Cells were thawed and washed twice with PBS, and then the cells were lyzed in RIPA buffer. SDS-PAGE was used to separate the proteins in the samples. The electrophoresed proteins were transferred onto a PVDF membrane using the semi-dry method (15 V, 35 min). After blocking with 5% milk for 1 h, the membranes were incubated with anti-adenosine A2b receptor (AAR-003, alomone labs) or anti-GAPDH (sc-32233, Santa Cruz Biotechnology) antibodies at 4°C overnight. Finally, the membranes were incubated with peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L) (JAC-711-035-152, Jackson, MS, United States) or peroxidase-conjugated AffiniPure donkey anti-mouse IgG (H + L) (JAC-715–035–150, Jackson, MS, United States) at room temperature for 1 h, and the blots were visualized with a Bio-Rad Imager (Bio-Rad, Hercules, CA, United States).
Peroxidase conjugated affinipure donkey anti rabbit igg h l
Manufactured by Jackson ImmunoResearch
Sourced in United States
Peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L) is a secondary antibody used to detect and visualize rabbit primary antibodies in various immunoassays. The antibody is conjugated with the enzyme peroxidase, which catalyzes a colorimetric reaction, allowing for the detection and localization of the target rabbit IgG.
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2 protocols using peroxidase conjugated affinipure donkey anti rabbit igg h l
1
Western Blot Analysis of Adenosine A2b Receptor
2
Smad3 Protein Expression Analysis
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1 × 106 T cells were treated with 0.1 μM, 1 μM, or 10 μM Galunisertib for 24 h in a 48-well plate, and DMSO was used as control. The cells were collected, washed with PBS, lysed by ultrasound, and boiled in a metal bath. The proteins were separated by SDS-PAGE and transferred onto a PVDF membrane by a semi-dry transfer method. The membrane was blocked with 5% BSA for 2 h and incubated with the anti-smad3 (EP823Y, Abcam, Cambridge, UK) or anti-GAPDH (sc-32233, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody at 4°C overnight. After incubating with Peroxidase-conjugated AffiniPure Donkey Anti-Rabbit IgG (H + L) (711-035-152, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or Peroxidase-conjugated AffiniPure Donkey Anti-Mouse IgG (H + L) (715-035-150, Jackson ImmunoResearch Laboratories) for 1 h at room temperature, the proteins were detected with a Bio-Rad ChemiDoc MP chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).
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