Anti tubulin antibody

For CLSM analysis, 0.5 × 10⁶ ds Red A. fumigatus conidia were applied to the apical surface and at several time points post infection as indicated in the Figure legends or Results, inocula were removed by gentle suction and the well was fixed using 4% paraformaldehyde solution for 10 minutes. After fixation the cells were treated as described above except staining the slides for mucin using rabbit anti-human MUC5B antibody (1:80; Abcam), followed by donkey anti-rabbit IgG (1:50 Biolegend). Again, actin localized to tight junctions was identified by using phalloidin-650 (1:20; Cell Signaling Technology). Cilia were stained using an anti-tubulin antibody (1:10; BD Pharmingen), and nuclei using Hoechst 33342 (Cell Signaling Technology). Images were captured using a Leica SP5 confocal laser scanning microscope and analysed using the LAS AF Lite or Imaris software. For each condition at least 50 cells were analyzed and used for quantifications.

Cultured cells were lysed in RIPA buffer containing 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM Na₃VO₄, 1 mM NaF and incubated for 10 min at 4°C. For whole cell lysate preparation lysates were subjected to sonication. The lysates were centrifuged at 10,000 rpm for 10 min. For immunoprecipitation 600 µg total cell lysate with 4 µg of specific antibodies was used. After 3 hours immunocomplexes were precipitated with A/G PLUS-agarose beads. Precipitates were washed 3 times in PBS buffer containing protease inhibitors and subjected to SDS-electrophoresis.
Isolation of cytosolic and nuclear fractions was performed as described [34] (link).
Antibodies against P-Chk-1, Chk-1, P-Chk2, γH2AX, H2AX, Phospho-ATM, ATM, Phospho-ATR, ATR, Histone H3 were from Cell Signaling. Antibodies for Chk-2 and P-Chk-2 (sc-16297-R) were from Santa Cruz. Anti-uPAR monoclonal antibody was from R&D Systems. Antibodies against 19S proteasome subunits were from Enzo Life Sciences. Anti-tubulin antibody was from BD Pharmingen. Alexa Fluor 488-conjugated chicken anti-rabbit antibody and Alexa Fluor 594-conjugated donkey anti-mouse antibody were from Life Technologies.
Western blotting images were acquired using VersaDoc Imaging system (Bio-Rad) and quantified using QuantityOne software (Bio-Rad). Expression of phosphorylated proteins was normalized to the level of total protein expression.