Anti rab14

Total protein from cells was extracted using RIPA buffer (Pierce, Rockford, IL, USA). The protein concentration was determined using the Bradford method (Pierce). Equal amounts of protein (40 μg) were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS_PAGE) and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in phosphate-buffered saline and Tween-20 for 1 h, membranes were incubated with anti-RAB14, anti-Akt, anti-p-Akt, anti-CCND1, anti-CDK2, or anti-Bax antibody (1 : 2000 dilution; Abcam, Hong Kong, China) as well as anti-GAPDH antibody (1 : 2000; Abcam) at 4°C overnight followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 5,000 dilution) at 37°C for 1 h. Immunoreactive bands were detected using the ECL Plus Detection kit (Pierce).

The total protein from cells was extracted using RIPA buffer (Pierce, Rockford, IL, USA). The protein concentration was determined using the Bradford method (Pierce). Equal amounts of proteins (40 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in phosphate-buffered saline (PBS)–Tween-20 for 1 h, the membranes were incubated with anti-RAB14, anti-Akt, anti-p-Akt, anti-CCND1, anti-CDK2, or anti-Bax-antibody (1:2000 dilution, Abcam, Hong Kong, China), as well as anti-GAPDH (1:2000, Abcam) antibody at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5000) at 37 °C for 1 h. Immunoreactive bands were detected using the ECL Plus Detection kit (Pierce, Rockford, IL, USA).

In this study, we used the following antibodies: goat polyclonal to Chlamydia trachomatis MOMP coupled to FITC (Abcam, ab30951, USA); rabbit polyclonal anti-RAB14 (Abcam, ab28639, USA); rabbit monoclonal anti-GM130 (Abcam, ab52649, USA); rabbit polyclonal anti- GOLGA5/Golgin-84 (Abcam, ab224040, USA); rabbit polyclonal anti-Akt (pan) (Cell Signaling, 4685, USA); rabbit polyclonal anti-phosphorylated Akt (Ser-473) (Cell Signaling, 4060, USA); rabbit polyclonal anti-AS160 (Affinity, AF7630, USA); rabbit polyclonal anti-phosphorylated AS160 (Ser-318) (Affinity, AF2317, USA); rabbit polyclonal anti-GAPDH (Cell Signaling, 2118, USA); goat anti-rabbit HRP-conjugated IgG (ABclonal, AS007, China), and goat anti-rabbit Cy3-labeled IgG (ABclonal, AS007, China). To activate Akt, cells were pretreated with SC79 (Beyotime, SF2730, China) for 30 min. To inhibit Akt, Akt Inhibitor VIII (iAkt) (Beyotime, SF2730, China) was added to the culture medium at the indicated post-infection (p.i.) time. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, France) was used as a control for SC79 and iAkt.