Proteins were extracted using RIPA lysis buffer (Biosharp, Shanghai, China) supplemented with protease inhibitors. The proteins were separated in 10% SDS-PAGE (Beyotime, Shanghai, China) and electrophoretically transferred to PVDF membrane (Milipore, USA). The membranes were incubated with primary antibodies: Actin(1:3000, #bs0061R, Bioss, Beijing, China), Musashi (1:1000, Bioss, Beijing, China), Snail (1:1000, #bs1371R, Bioss, Beijing, China), Notch1 (1:100, #AF5307, Affinity Biosciences, OH, USA), Hes1 (1:200, #BM4488, Boster Biological Technology Co. Ltd., Wuhan, China), and JAG1 (1:500, #bs-1448R, Bioss, Beijing, China) at 4 °C overnight. HRP-conjugated secondary antibodies (1:5000, #bs0295M, Bioss, Beijing, China)were used for 1 h at 37 °C. The protein bands were observed using ECL reagents (Milipore, USA). Protein immunoreactivity derived from vehicle groups (LV-NC) was normalized to 1.0. Values of LV-miR-153 groups were normalized according to vehicle groups (LV-NC).
Af5307
Manufactured by Affinity Biosciences
Sourced in United States
The AF5307 is a lab equipment product manufactured by Affinity Biosciences. It is a scientific instrument designed for laboratory use. The core function of the AF5307 is to perform specific tasks in a controlled and precise manner within a laboratory setting. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sds page, by Beyotime (1 mentions)
Bs 0295m, by Bioss Antibodies (1 mentions)
Bs 1371r, by Bioss Antibodies (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Bs 1448r, by Bioss Antibodies (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Biosharp (1 mentions)
Ab85763, by Abcam (1 mentions)
Ab65297, by Abcam (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
3 protocols using af5307
1
Analyzing Notch Pathway Proteins in Cells
2
Immunohistochemical Analysis of Notch Pathway
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Formalin-fixed paraffin-embedded tissues were sectioned at 4 µm thickness using routine deparaffinization and rehydration procedures. The sections were stained as previously described (Zhang et al. 2016a) . The primary antibodies used in these experiments were JAG1 (#70109, 0.5 μg/mL, CST, USA), NOTCH1 (#3608, 4.2 μg/mL, CST) and N1ICD (AF5307, 5 μg/ mL, Affinity). Sections incubated without the primary antibody served as negative controls, while sections obtained from squamous cell lung cancer, cervical squamous carcinoma and prostatic carcinoma that positively expressed JAG1, NOTCH1 and N1ICD respectively were used as positive controls as shown in Supplementary Fig. 1 (see section on supplementary data given at the end of this article).
3
Quantifying Protein Expression in Tissues
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Equal amounts of proteins extracted from tissue samples (30 µg) and cell samples (90 µg) were loaded and subjected to 8% SDS-PAGE, and the following steps were performed as previously described (Zhang et al. 2016a) . The primary antibodies used in these experiments were JAG1 (ab85763, 1.25 µg/mL, Abcam), NOTCH1 (ab65297, 1.6 µg/mL, Abcam), VEGF (19003-1-AP, 0.73 µg/mL, Proteintech), MMP9 (10375-2-AP, 0.55 µg/mL, Proteintech), N1ICD (AF5307, 1 µg/mL, Affinity, USA) and GAPDH (AF7021, 1 µg/mL, Affinity). Optical densities (ODs) were measured by ImageJ (Rawak Software, Inc. Germany). Expression was calculated as the ratio of the target OD to the GAPDH OD in each case.
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