Interaction with and stabilization of microtubules by BtpA against Nocodazole-induced depolymerization is WxxxE-dependent. HeLa cells were transfected with plasmids expressing GFP-BtpA (C, D), GFP-BtpA WxxxE variants (E-L), or I226S (M), respectively. Sixteen hours after transfection cells were either left untreated (A, C, E, G, I, K) or treated with 1 μg/ml nocodazole for 30 min (B, D, F, H, J, L, M). Nocodazole-treated and control cells were fixed and stained with mouse anti β-tubulin antibody, followed by detection with anti-mouse Texas Red, and analysed by fluorescence microscopy. (A) Fluorescent image of tubulin-stained non-transfected control cells (B) Fluorescent image of non-transfected, nocodazole-treated cells. (C-M) Individual green and red fluorescent images and overlay image of HeLa cells expressing (C-D) GFP-BtpA, (E-F) GFP-BtpA W213A, (G-H) GFP-BtpA W213S, (I-J) GFP-BtpA W213F, (K-L) GFP-BtpA E217A, (M) GFP-BtpA I226S. Arrow heads indicate individual cells showing inhibition of nocodazole-induced microtubule depolymerization by expression of (D) GFP-BtpA, (F) GFP-BtpA W213A, and (M) and I226S, in contrast to non-transfected cells. Arrows indicate nocodazole-induced microtubule depolymerization in individual cells expressing (H) GFP-BtpA W213S, (J) GFP-BtpA W213F, (L) GFP-BtpA E217A. Scale bar, 25 μm indicated in A applies to all individual green and red fluorescent images. The overlay images are close ups of individual cells in the corresponding red and green fluorescent images. Data are representative of 3 or more independent experiments.