A: BCCIP knockdown reduces microtubule acetylation. Control and BCCIP knockdown HeLa cells or MEF control and MEF BCCIP knockout cells were lysed and blotted for the stable microtubule marker, acetyl-tubulin.
B: Levels of acetylated-tubulin in control and BCCIP knockdown cells. HeLa cells expressing BCCIP-shRNA (weak green, un-arrowed) were mixed with control cells (strong green, arrowed), and stained for stained for BCCIP and acetyl tubulin.
C: The reduced acetyl-tubulin in BCCIP deficient cell can be rescued by RNAi-resistant BCCIPα. RNAi-resistant YFP-BCCIPα, YFP-BCCIPβ, and YFP were re-expressed in BCCIP knockdown cells. These YFP-positive cells were mixed with non-transfected knockdown or control cells (YFP-negative cells). Total fluorescent intensity of acetyl tubulin in BCCIP knockdown (YFP-negative) and BCCIP re-expressed (YFP positive) cells on the same staining slide was quantified. Shown are relative intensities of acetyl-tubulin in wild type cells (first column from left), BCCIP knockdown cells (2nd column from left), and the knockdown cells expressing different YFP-tagged proteins. Representative images of rescue can be viewed in Supplement Figure S4.