Western Blot Analysis of SAMHD1 Expression

For analysis of the levels of expression of total and phosphorylated SAMHD1 in siRNA-transfected iDCs or MDMs, as well as in MDM-derived MGCs, cells (10⁶) were lysed in 100 μl of reducing Laemmli sample buffer (2×) supplemented with PhosSTOP (phosphatase inhibitor cocktail tablets; Sigma-Aldrich) and cOmplete (EDTA-free protease inhibitor cocktail; Sigma-Aldrich) and then boiled at 96°C for 10 min. Cell lysates were then resolved by SDS-PAGE as described previously (64 (link)) and analyzed by Western blotting using anti-SAMHD1 (Abcam; ab67820), anti-phospho-SAMHD1 (Cell Signaling; catalog no. 89930), anti-HIV-1 p24 (Abcam; ab9071), and anti-β-actin (Sigma-Aldrich; clone AC-15) antibodies.

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Xie M., Leroy H., Mascarau R., Woottum M., Dupont M., Ciccone C., Schmitt A., Raynaud-Messina B., Vérollet C., Bouchet J., Bracq L, & Benichou S. (2019). Cell-to-Cell Spreading of HIV-1 in Myeloid Target Cells Escapes SAMHD1 Restriction. mBio, 10(6), e02457-19.

Publication 2019

PhosSTOP ab67820 anti-β-actin

Actin Antibodies Cell Clone Edta Hiv p24 Idcs Laemmli buffer Mdms Phosphatase Protease inhibitor Samhd1 Sds page Sirna

Corresponding Organization :

Other organizations : Université Paris Cité, Inserm, Centre National de la Recherche Scientifique, Institut Cochin, Délégation Paris 5, Sorbonne Paris Cité, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Institut de Pharmacologie et de Biologie Structurale, Université Toulouse III - Paul Sabatier

Top 5 similar protocols

Variable analysis

independent variables

SiRNA transfection

dependent variables

Levels of expression of total SAMHD1
Levels of expression of phosphorylated SAMHD1
Levels of HIV-1 p24

control variables

Cell type (iDCs, MDMs, MDM-derived MGCs)
Cell number (10^6)
Lysis buffer (reducing Laemmli sample buffer (2×) supplemented with PhosSTOP and cOmplete)
Boiling time (10 min at 96°C)
SDS-PAGE and Western blotting

controls

Anti-β-actin antibody (as a loading control)

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