Protein Extraction and Quantification

Protein extraction and assay were as previously described (33 (link), 34 (link), 64 (link), 65 (link)). Cells were lysed with radioimmunoprecipitation (RIPA) buffer (Sigma, Cat. # R0278) supplemented with protease and phosphatase inhibitor cocktails (Sigma, Cat. # P8340 and P0044, respectively). The lysates were sonicated twice for 30 seconds at 25% output power with a Sonicator ultrasonic processor (Misonix, Inc., Farmingdale, NY) and centrifuged (14,000 g; 4°C) for 30 min. Total protein levels were determined by the bicinchoninic acid assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA, Cat.# 23228 and Cat.# 1859078).

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Balan I., Aurelian L., Williams K.S., Campbell B., Meeker R.B, & Morrow A.L. (2022). Inhibition of human macrophage activation via pregnane neurosteroid interactions with toll-like receptors: Sex differences and structural requirements. Frontiers in Immunology, 13, 940095.

Publication 2022

RIPA) buffer phosphatase inhibitor cocktails Sonicator ultrasonic processor BCA

Assay Bicinchoninic acid Buffer Cells Phosphatase Protease Protein Radioimmunoprecipitation Seconds Ultrasonic

Corresponding Organization : University of North Carolina at Chapel Hill

Other organizations : Stanford University, Sage Therapeutics (United States)

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Total protein levels

control variables

Protein extraction and assay procedures as previously described (references 33, 34, 64, 65)
RIPA buffer supplemented with protease and phosphatase inhibitor cocktails
Sonication parameters (twice for 30 seconds at 25% output power)
Centrifugation conditions (14,000 g; 4°C for 30 min)

controls

Positive control: Not specified
Negative control: Not specified

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