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. Author manuscript; available in PMC: 2021 May 19.
Published in final edited form as: Methods Mol Biol. 2019;1982:191–229. doi: 10.1007/978-1-4939-9424-3_12

Fig. 5.

Fig. 5

Examples of using positive and negative control lysates for the validation of NOX4 antibodies. Two laboratories tested several NOX4 antibodies from Table 8. Left column: NOX4 antibodies tested in the laboratory of Dr. J. D. Lambeth. Right column: NOX4 antibodies tested in the laboratory of Dr. J. Doroshow. Left column: Identical PVDF immunoblots consisting of 10 μg of the following non-heated cell line lysates. Lane (1) non-transfected HEK 293 cells, lane (2) HEK 293 cells stably transfected with pcDNA3.1-NOX4, untagged (a kind gift from Dr. R. Brandes (Goethe-University Frankfurt), lane (3) HEK 293 cells stably transfected with pcDNA3.1-NOX1, untagged (also a gift from Dr. Brandes). The blots were probed with the following NOX4 antibodies from Table 8 (a) Dr. A. Shah’s NOX4 rabbit pAb, 1:2000 (a kind gift from Dr. A. Shah, University College London); (b) Dr. J. Doroshow’s NOX4 rabbit mAb, clone 47–6, 1 μg/mL (1:1000); (c) NOVUS NOX4 rabbit pAb #NB110–58849 Lot F1 (1:1000, 1 μg/mL); (d) green, Dr. J. D. Lambeth’s NOX1 E39 mAb, 1:1000, as a positive control for NOX1; red, Dr. Shah NOX4 rabbit pAb, 1:2000. (e) β-actin mouse mAb (Sigma-Aldrich), 1:10,000. Blocking buffer, 5% nonfat dry milk in TBST; antibody diluent, LI-COR Odyssey blocking buffer +0.1% Tween-20; primary antibodies incubated overnight; secondary antibodies (HRP-conjugated goat anti-rabbit or anti-mouse), 1 h. ECL reagent: SuperSignal West Pico, 1 s. Right column: identical nitrocellulose membranes consisting of 40 μg of the following cell lysates. Lane (1) non-transfected HEK 293 cells, lane (2) HEK 293 transfected with pCMV6-empty vector, lane (3) HEK 293 stable clone (WT1) overexpressing N-terminal tagged-Myc-DDK-NOX4, lane (4) HEK 293 stable clone (WT2) overexpressing N-terminal tagged-Myc-DDK-NOX4. The following antibodies from Table 8 were tested: (f) Dr. P. Jansen-Dürr’s NOX4 mAb 1:500; (g) Millipore NOX4 pAb (#ABC549), 1:500; (h) Abcam NOX4 antibody (ab133303), 1:1000; (i) anti-Myc antibody; (j) anti-vinculin antibody. Conditions: similar to left panel, except the antibody was diluted in 5% milk in TBST and the ECL reagent was ECL Plus reagent (GE Healthcare)