Purified Mouse Anti-p62 Ick ligand Clone 3/P62 LCK LIGAND (RUO)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For WB, lyse cells in on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors	Incubate primary Ab overnight at 4°C

Upstream tips
For WB, lyse cells in on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors
Protocol tips
Incubate primary Ab overnight at 4°C

Publication protocol

Western blotting
SH‐SY5Y cells were harvested with trypsin and lysed on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors. Protein lysates (20 μg) were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to Hybond P (GE Healthcare, Little Chalfont, UK). Blots were probed with antibodies against Nrf2 (ab31163; Abcam, Cambridge, UK), p62 (610833; BD Biosciences, Oxford, UK), prohibitin 1 (2426; Cell Signaling Technology, Beverly, MA, USA), cytochrome oxidase (COX) subunit IV (ab14744; Abcam), LC3B (2775; Cell Signaling Technology), cathepsin D (ab6313; Abcam), TOM20 (FL‐145; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TFEB (ab2636; Abcam), DDK (clone 4C5; Origene), lamin A (ab26300; Abcam), PINK1 (BC100‐494; Novus Biologicals, Cambridge, UK), PGC‐1α (NBP1‐04676; Novus Biologicals) or β‐actin (ab82618; Abcam). Bands were detected with respective horse radish peroxidase‐linked secondary antibodies (Dako, Carpinteria, CA, USA) and enhanced chemiluminescence (Pierce). Density of bands was determined using ImageJ software (NIH, Bethesda, MD, USA) or Image Lab software 5.1 (Bio‐Rad Laboratories, Hercules, CA, USA).

Immunofluorescence
Following treatment with CCCP, SH‐SY5Y cells on coverslips were fixed in 3.7% paraformaldehyde. For detection of p62, cells were permeabilised with glacial methanol, blocked in 2% goat serum in PBS/0.1% Triton X‐100 and incubated with antibodies against p62 (610833; BD Biosciences) and TOM20 (FL‐145; Santa Cruz Biotechnology). For detection of Nrf2 or TFEB, cells were permeabilised with PBS/0.1% Triton X‐100, blocked with 2% goat serum and 2% bovine serum albumin in PBS/0.1% Triton X‐100, and incubated with Nrf2 antibody (ab31163; Abcam) or DDK antibody (see above) overnight at 4°C. Respective fluorescent secondary antibodies (alexa fluor 488, 594; Invitrogen, Carlsbad, CA, USA) were used for detection and coverslips mounted in citifluor containing 4′,6‐diamidino‐2‐phenylindole.

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Manufacturer protocol

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