Mouse anti γ tubulin clone gtu 88

Commercial antibodies used were mouse anti-γ-tubulin (clone GTU-88, 1/5000, Sigma), anti-acetylated α-tubulin (^Actub) (clone 6-11B-1, 1/10000, Sigma), anti-rhodopsin (clone 4D2, 1/100), anti-β–actin (clone AC-15, 1/30000, Sigma) and anti-β-tubulin (clone 2-28-33, 1/2000, Sigma), anti-EHD3 (1/1000, Abnova for IF and 1/100, Abcam, 1/500, Proteintech for WB), rabbit anti-pericentrin (1/5000, Novus Biologicals, 1/5000), anti-EHD2 (1/1000, Abcam), anti-EHD4 (1/1000, Abcam), anti-GFP (1/50, Abcam), anti-HA HRP (1:1000, Roche), anti-CEP290 (1/200, Bethyl), anti-RPGRIP1L (1/200, Proteintech), anti-IFT20 (1/200, Proteintech), anti-TMEM67 (1/200, Proteintech), anti-SNAP29 (1/1000 for WB Proteintech and 1/200 for IF from Dr. Andrew Peden) and goat anti-CEP164 (1/500, Santa Cruz). Rabbit anti-Rab11a (1/300) was a gift from Dr. Jim Goldenring and rabbit anti-CP110 (1/500) was a gift from Dr. Monica Diaz. Polyclonal rabbit anti-EHD1 (1/1000) has been previously described^{33 (link)}. Fluorescent secondary antibodies and phalloidin (1/50) were from Life Technologies (Molecular probes, Eugene, OR). Nuclei were labeled with Hoechst (Molecular probes, Eugene, OR).

Tubulin staining was performed using mouse anti-γ tubulin, clone GTU-88 (Sigma). The anti-DYNLT1 monoclonal antibody was from Millipore. The chlamydia antibodies used were polyclonal rabbit anti-C. trachomatis L2 EB. Rabbit anti-peptide antibodies against Inc850 (TVKDSFLKKARRERFLA) were prepared commercially by Quality Controlled Biochemicals, Inc (Hopkinton, MA). C. trachomatis-infected cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS and stained for immunofluorescence microscopy as previously described [17 (link)].
Images were acquired on a Nikon Eclipse 80i microscope using a 60× 1.4 NA oil immersion objective (Nikon, USA) or a Zeiss LSM 510 Meta laser confocal scanning microscope using a 63×, 1.4 NA oil objective (Carl Zeiss MicroImaging, Inc., Maple Grove, MN)
To determine distance from the chlamydial inclusion to centrosomes, C. trachomatis-infected HeLa cells at 18 hr post-infection were fixed and stained with anti-chlamydia sera and anti-γ tubulin for centrosomes. Images were acquired and merged and the closest distance from the inclusion membrane to the nearest centrosome was determined using an NIS-Elements AR software package (Nikon).