HEK293 cells (ATCC CRL‐1573) were grown under standard conditions and transfected with FLAG (DDK)‐tagged plasmids expressing human or mouse PNPLA2 or PNPLA3, or VHH‐FLAG‐Halo (Table S1 ). Cells were transfected using Fugene HD (Promega) according to the instructions from the vendor. After 48 hours, cells were lysed and western blot experiments performed as detailed in the Supporting Materials and Methods . The following primary antibodies were used: sheep polyclonal anti‐human PNPLA3 (1:1000, AF5208; R&D Systems, Minneapolis, MN, USA), mouse monoclonal anti‐FLAG M2 (1:1000, F1804; Sigma Aldrich, USA), and rabbit polyclonal anti‐alpha tubulin (1:200, ab4074; Abcam, UK). Membranes were washed and incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies anti‐rabbit (1:3000, 7074S; Cell Signaling Technologies, Inc, USA), anti‐mouse (1:3000, 7076S; Cell Signaling Technologies), or anti‐sheep (1:2000, ab6900; Abcam). Western blots were visualized with chemiluminescent HRP substrate (WBLUR0500; Millipore, USA), using the ChemiDoc MP imaging system (Bio‐Rad, USA).
Ab6900
Manufactured by Abcam
Sourced in United States, Netherlands
Ab6900 is a lab equipment product offered by Abcam. It is a core piece of laboratory equipment used for general purpose applications. The detailed specifications and intended use of this product are not available in the concise format requested.
Automatically generated - may contain errors
Lab products found in correlation
Ab4074, by Abcam (1 mentions)
Wblur0500, by Merck Group (1 mentions)
Af5208, by R&D Systems (1 mentions)
F1804, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Fugene hd, by Promega (1 mentions)
Dm001, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
2 protocols using ab6900
1
PNPLA2 and PNPLA3 Overexpression in HEK293 Cells
2
Western Blot Analysis of UCP1 in BAT
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Proteins were isolated from snap frozen brown adipose tissue (BAT) by lysing tissues with RIPA buffer supplemented with protease inhibitors, after which the protein lysate was collected. Western blot was performed loading 4 µL of 0.2 µg/mL lysate on the Wes apparatus (ProteinSimple, Wiesbaden, Germany), according to the manufacturer’s protocol. The following primary and secondary antibodies were used: UCP1 (Sigma-Aldrich, U6382, 1:20), GAPDH (Santa Cruz Biotechnology, sc25778, 1:50), HRP anti-rabbit (ProteinSimple, DM001) and HRP anti-sheep (Abcam, Amsterdam, the Netherlands, ab6900, 1:100).
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