Total cell lysates were prepared following published methods [42 (link)]. Membrane fraction of lysates were prepared with MEM-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). Immunoblotting was performed as described previously [43 (link)]. Membranes were blocked with 5% skim milk in Tris-buffered saline with Tween (TBST) or Blocking-One reagent (Nacalai Tesque, Kyoto, Japan). Primary antibodies are listed in Supplementary Table S3 . Primary antibodies were diluted with TBST or Can Get Signal 1 (Toyobo, Osaka, Japan). Data shown are representative of at least two experiments with similar results, unless otherwise mentioned.
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Membrane Protein Extraction and Immunoblotting
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Variable analysis
independent variables
- Methods for preparing total cell lysates [42]
- Membrane protein extraction using MEM-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific)
- Protein expression levels measured by immunoblotting
- Blocking of membranes with 5% skim milk in Tris-buffered saline with Tween (TBST) or Blocking-One reagent (Nacalai Tesque, Kyoto, Japan)
- Dilution of primary antibodies with TBST or Can Get Signal 1 (Toyobo, Osaka, Japan)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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