KGN cells seeded onto glass coverslips in six-well plates at a density of 5 × 104 were transfected with plasmids for expression of FLAG-tagged AMH and treated with CTX, GnRHa, or CTX and GnRHa. After 48 h, immunofluorescence staining assays were carried out, as previously described39 (link), with primary antibodies for AMH (ab103233, Abcam, Shanghai, China) and GRP78 (AF5366, Affinity, Jiangsu, China), and secondary goat anti-rabbit antibodies conjugated to FITC or Alexa Fluor 594 (Proteintech). Images were visualized and captured with a TCS SP5 confocal microscope Leica (Shanghai, China).
Af5366
Manufactured by Affinity Biosciences
Sourced in China
AF5366 is a laboratory instrument designed for performing high-performance liquid chromatography (HPLC) analysis. The core function of this product is to separate, identify, and quantify components in a mixture through the process of liquid chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab103233, by Abcam (1 mentions)
Alexa fluor 594, by Proteintech (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Ab214421, by Abcam (1 mentions)
Bm1623, by Boster Bio (1 mentions)
Ab229912, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
3 protocols using af5366
1
AMH Expression and Localization by IF
2
Investigating ER Stress Pathways in Immune Checkpoint Regulation
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Cells were collected and lysed in the whole-cell lysate (containing phenylmethylsulfonyl fluoride and a phosphatase inhibitor). Equal amounts of cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking in 5% skimmed milk for 1 h at 37°C, membranes were incubated overnight at 4°C with the following primary antibodies: anti-GRP78 (AF5366, Affinity, China, 1 : 1000), anti-PERK (ab229912, Abcam, UK, 1 : 1000), anti-phospho-PERK (p-PERK, DF7576, Affinity, China, 1 : 2000), anti-CHOP (BF8081, Affinity, China, 1 : 1000), anti-programmed cell death receptor 1 (PD-1, ab214421, Abcam, UK, 1 : 1000), and anti-mucin domain 3 (TIM-3, D3M9R, Cell Signaling Pathway, USA, 1 : 1000), and anti-GAPDH (BM1623, Boster Biological Technology, China, 1 : 5000) was used as a control. Following the primary incubation, the membranes were incubated with horseradish peroxidase-conjugated goat antimouse IgG secondary antibody (ZJ2020, Biygot, China, 1 : 10000) at room temperature for 2 h. After washing the membrane with TBST for 45 min, the protein signal was detected using ECL Plus Hypersensitive Luminescence solution (AR1197, Boster, China).
3
Western Blot Analysis of Protein Expression
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RIPA buffer (Beyotime Institute of Biotechnology) was added to the treated cells to obtain the total protein samples. Protein concentration was then determined by BCA kit (Beyotime Institute of Biotechnology). Then, 10% SDS-PAGE was prepared, subjected to electrophoresis and transferred to PVDF membranes. Blots were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with antibodies separately. The membranes were incubated in the primary antibody overnight at 4°C, washed by TBST containing 0.1% Tween-20 and soaked in the secondary antibody for 1 h at room temperature. The primary antibodies used were as follows: anti-β-actin (1:10,000; Abcam; cat. no. ab179467), anti-Sig-1R (1:500; ProteinTech Group, Inc. 15168-1-AP), anti-CHOP (1:1,000; Affinity Biosciences, DF6025), anti-glucose-regulated protein 78 (GRP78) (1:1,000; Affinity Biosciences, AF5366), anti-Bax (1:1,000; Affinity Biosciences, AF0120), anti-Bcl-2 (1:1,000; BIOSS, bs-4563R) and anti-cytochrome c (1:5,000; Abcam, ab133504). The secondary antibody used was the goat anti-rabbit conjugated with HRP (1:10,000; Abcam, ab6721). The immunoreactive bands were visualized with an automatic chemiluminescence image analysis system (Tanon 5200, China) The Bandscan 4.3 software (Glyko) was used to analyze the gray value of the protein.
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