The snap-frozen tissues were subjected to homogenization in 250μL of lysis buffer as previously described. Samples (30 μg of protein for each condition) were transferred onto PVDF membranes and then incubated with antibodies (Li et al., 2017 (link)). The following were used as primary antibodies: caspase 3 (ab323519, Abcam), myeloid differentiation factor 88 (MyD88) (SC74532, Santa Cruz), toll-like receptor 4 (TLR4) (AF7017, Affinity), ZO-1 (AF 5145, Affinity), occludin (DF7504, Affinity), and NF-κB(ab16502, Abcam). Immunoreactive bands were revealed using a 1:10,000 dilution of secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit IgG, BE0101, Bioeasy; goat anti-mice IgG, BE0102, Bioeasy). The blots were re-probed with antibodies against β-actin (EASYBIO) and GAPDH (Bioworld) to ensure equal loading and transfer of proteins. All critical blots and immunoprecipitation experiments were repeated at least three times.
Ab323519
Manufactured by Abcam
Ab323519 is a laboratory equipment product. It serves as a core functional component for scientific research and analysis.
Automatically generated - may contain errors
2 protocols using ab323519
1
Western Blot Analysis of Signaling Proteins
2
Protein Expression Analysis of Tissue Samples
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The snap-frozen tissues were subjected to homogenization in 250ml of lysis buffer as previously described. Samples (of 30 mg of protein for each condition) were transferred onto PVDF membranes and then incubated with antibodies [24] . The following were used as primary antibodies: caspase 3 (ab323519, Abcam), myeloid differentiation factor 88 (MyD88) (SC74532, Santa Cruze), toll-like receptor 4 (TLR4) (AF7017, A nity), ZO-1 (AF 5145, A nity), occludin (DF7504, A nity), and NF-κB(ab16502, Abcam). Immunoreactive bands were revealed using a 1:10,000 dilution of secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit IgG, BE0101, Bioeasy; goat anti-mice IgG, BE0102, Bioeasy). The blots were re-probed with antibodies against β-actin (EASYBIO) and GAPDH (Bioworld) to ensure equal loading and transfer of proteins. All critical blots and immunoprecipitation experiments were repeated at least three times. Selected blots were quanti ed with ImageJ software.
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