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Blots were subsequently blocked and re-probed with a mouse anti tubulin antibody (0.15 μg/ml; Abcam, Cambridge, UK, ab11323).
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Western blots were prepared by using nitrocellulose membranes (PROTRAN, Schleicher & Schuell, Inc., Keene, NH) which were subsequently blocked using 5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4.
This was subsequently blocked by a bulky amino acid substitution.
The blots were subsequently washed with TBST and incubated with respective secondary antibodies.
Blots were subsequently analyzed by Quantity One software (Biorad, Hercules, USA).
In some case the blots were subsequently stained with Ponceau red to directly visualize the proteins.
The blots were subsequently incubated with the corresponding peroxidase-conjugated IgG antibody.
The blots were subsequently stripped and reprobed with tubulin antibody.
Blots were subsequently probed with Clean-Blot IP-horseradish peroxidase conjugate (Thermo Scientific).
The blots were subsequently reprobed with Vinculin to ascertain equal protein loading.
Blots were subsequently incubated with IRDye 680 or 800 labelled secondary antibodies for 1 h.
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