Western Blot Analysis of Autophagy Markers

As previously mentioned^{48 (link)}, proteins were isolated and electroblotted. The membranes were blocked for 1 h with 5% BSA (ZSGB-BIO, Beijing, China) and incubated overnight at 4℃ with the primary antibodies (anti-Beclin-1, 1:1000dilution; anti-LC3, 1:2000 dilution; anti-RUNX2, 1:1000 dilution; anti-P62/SQSTM1, 1:5000 dilution, all from Abcam, MA, USA). After washing, membranes were incubated for 1 h with the secondary horseradish peroxidase antibody (Solarbio, Beijing, China). Finally, the intensity of the bands was determined using the Bio-Rad VersaDoc imaging system and the ECL kit (Solarbio, Beijing, China).

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Dang H., Chen W., Chen L., Huo X, & Wang F. (2023). TPPU inhibits inflammation-induced excessive autophagy to restore the osteogenic differentiation potential of stem cells and improves alveolar ridge preservation. Scientific Reports, 13, 1574.

Publication 2023

anti-Beclin-1 anti-LC3 anti-RUNX2 anti-P62/SQSTM1 ECL kit

Antibodies Antibody Beclin 1 Dilution Horseradish peroxidase Membranes Proteins Runx2

Corresponding Organization :

Other organizations : Southwest Medical University, Dalian Medical University

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Variable analysis

independent variables

Blocking of membranes for 1 h with 5% BSA
Incubation of membranes overnight at 4℃ with primary antibodies (anti-Beclin-1, anti-LC3, anti-RUNX2, anti-P62/SQSTM1)
Incubation of membranes for 1 h with secondary horseradish peroxidase antibody

dependent variables

Intensity of the protein bands

control variables

Protein isolation and electroblotting

controls

Positive control: Not specified
Negative control: Not specified

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