Immunoprecipitation and Western Blot Analysis

Immunoprecipitation and Western blot assays were performed according to the previously described procedures [4 (link)]. Antibodies Runx2 (Abcam, UK, 1:1000), ALP (Abcam, UK, 1:2000), SM22ɑ (Abcam, UK, 1:1000), p16 (Proteintech, USA, 1:500), p21 (Cell signaling, USA, 1:1000), LC3-II (Abcam, UK, 1:500), SQSTM1 (Abcam, UK, 1:1000), Atg10 (Abcam, UK, 1:1000), Bhlhe40 (Proteintech, USA, 1:500), GAPDH (Abcam, UK, 1:4000), and β-actin (Proteintech, USA, 1:2000) were used in this study. HA-VSMCs were transfected as indicated and lysed in an immunoprecipitation buffer. The lysates were centrifuged for 20 min. Supernatants were incubated with specific antibodies overnight at 4℃ and protein A/G Agarose beads for 4 h. The immunoprecipitates were washed three times with PBS, and then analyzed via SDS-PAGE. The protein bands were visualized using ECL-Plus Western blot detection kit (Amersham BioSciences, UK).

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Li S., Ni Y., Li C., Xiang Q., Zhao Y., Xu H., Huang W., Wang Y., Wang Y., Zhan J, & Liu Y. (2022). Long noncoding RNA SNHG1 alleviates high glucose-induced vascular smooth muscle cells calcification/senescence by post-transcriptionally regulating Bhlhe40 and autophagy via Atg10. Journal of Physiology and Biochemistry, 79(1), 83-105.

Publication 2022

Runx2 LC3-II SQSTM1 Atg10 Bhlhe40 GAPDH β-actin ECL-Plus Western blot detection kit

Actin Agarose Antibodies Buffer Gapdh Immunoprecipitation Protein Protein a Protein g Runx2 Sds page Western blot Western blot assays

Corresponding Organization :

Other organizations : Second Xiangya Hospital of Central South University, Central South University

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Variable analysis

independent variables

Transfection of HA-VSMCs

dependent variables

Protein expression levels of Runx2, ALP, SM22ɑ, p16, p21, LC3-II, SQSTM1, Atg10, and Bhlhe40

control variables

Immunoprecipitation and Western blot assays were performed according to the previously described procedures [4]
Antibodies used: Runx2 (Abcam, UK, 1:1000), ALP (Abcam, UK, 1:2000), SM22ɑ (Abcam, UK, 1:1000), p16 (Proteintech, USA, 1:500), p21 (Cell signaling, USA, 1:1000), LC3-II (Abcam, UK, 1:500), SQSTM1 (Abcam, UK, 1:1000), Atg10 (Abcam, UK, 1:1000), Bhlhe40 (Proteintech, USA, 1:500), GAPDH (Abcam, UK, 1:4000), and β-actin (Proteintech, USA, 1:2000)
Immunoprecipitation protocol: HA-VSMCs were lysed in an immunoprecipitation buffer, centrifuged, and supernatants were incubated with specific antibodies overnight at 4°C and protein A/G Agarose beads for 4 h. The immunoprecipitates were washed three times with PBS, and then analyzed via SDS-PAGE. The protein bands were visualized using ECL-Plus Western blot detection kit (Amersham BioSciences, UK)

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