Immunoblotting Analysis of Cell Signaling

The total protein of cells was prepared as previously described [32 (link)]. Sample proteins (30 μg) were separated via SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 5% non-fat milk. After incubation with the indicated antibodies, the membranes were washed and incubated with the secondary antibodies. Immunoreactivity was detected using ECL reagents. The following primary antibodies were used in this study: β-actin antibody (AP0060, Bioworld, Nanjing, China), TLR4 antibody (sc-293072, Santa Cruz Biotechnology, Paso Robles, CA, USA), Cav-1 antibody (ab192869, ABCAM, Cambridge, UK), Ras homolog family member A (RhoA) antibody (2117S, Cell Signaling Technology, Danvers, MA, USA), p65 antibody (10745-1-AP, Cell Signaling Technology), p-p65 antibody (3033S, Cell Signaling Technology), IκBα antibody (4814S, Cell Signaling Technology), and p-IκBα antibody (2859S, Cell Signaling Technology).

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Yuan H., Hou Q., Feng X., Zhang Y., Yang F., Ge R, & Li Q. (2022). 5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone Inhibits LPS-Induced Vascular Inflammation by Targeting the Cav1 Protein. Molecules, 27(9), 2884.

Publication 2022

β-actin antibody (AP0060, TLR4 antibody (sc-293072, p-p65 antibody IκBα antibody p-IκBα antibody

Actin Antibodies Antibody Family member Iκbα Membranes Milk Nitrocellulose Proteins Rhoa Sds page

Corresponding Organization : Shanxi Medical University

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Variable analysis

independent variables

Antibodies used (β-actin, TLR4, Cav-1, RhoA, p65, p-p65, IκBα, p-IκBα)

dependent variables

Protein expression levels

control variables

Protein sample amount (30 μg)
SDS-PAGE separation and protein transfer to nitrocellulose membranes
Blocking with 5% non-fat milk
Incubation with primary and secondary antibodies
ECL reagents for detection of immunoreactivity

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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