Antibody against α tubulin

Mosquito carcasses, 30 each, were homogenized by pellet pestle in 100 μl of lysis buffer [42 (link)]. Aliquots of mosquito protein samples were resolved using SDS-polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes (Merck Millipore). Then, membranes were blocked with Starting Block T20 (PBS) Blocking Buffer (Thermo Fisher Scientific) and incubated with the primary antibody against HPX8C (1:3000). Following three washes with TBS containing Tween-20, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (Sigma, 1:10000). After washing four times with TBS containing Tween-20, membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate reagent for 5 min (Thermo Fisher Scientific) before visualization. The antibody against α-tubulin (Cell Signaling Technology) was used as the loading control.

Caco-2 cells (6.7 × 10⁴) were seeded in 24-well plates and infected with HCMV isolate. After one hour the inoculum was discarded and the cells were washed for three times with PBS. At 0, 36, 96, and 168 h p.i. cells extracts (pooled triplicates) were solubilized in 4 × sample buffer (4% (v/v) ß-mercaptoethanol, 0.01% (w/v) bromophenol blue, 4% (w/v) glycerol, 4% (w/v) SDS, 0.2 M Tris-HCl (pH 6.8)) prior to separation on 10% (w/v) SDS-PAGE. Proteins were transferred to nitrocellulose sheets and subjected to Western blot analysis as described previously [35 (link)]. The antibody CCH2/DDG9 (Dako-Agilent Technologies; 1:500) specific for IE1/2 were used as the primary antibodies. For detection of primary antibody binding, horseradish peroxidase-conjugated anti-mouse F(ab′)2 fragments (1:5000 in PBS with 0.3% BSA; Abcam, Cambridge, UK). The membranes were reprobed with an antibody against α-tubulin (1.5000; Cell Signaling Technology) and HRP-conjugated anti-mouse F(ab′)2 to verify equal loading. Detection of protein bands was performed using ECL (Super Signal West Pico) reagent as recommended by the supplier (Pierce; Thermo Fisher Scientific, Henningsdorf, Germany). As a control, we used HELF Fi301 after 168 h p.i.

BMT (#B3023), TMZ (#T2577), propidium iodide (PI, #P4864), and MTT (#M2128) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat# 12430-054) and Penicillin/streptavidin (Cat# 15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from invitrogen (Carlsbad, CA). Anti-phospho-NKCC1(pThr206) antibody, anti-phospho-SPAK/OSR1 (pSer383 SPAK/pSer325 OSR1) antibody, and anti-total-SPAK/OSR1 (tSPAK/tOSR1) antibody were developed by Dr. Yang (Taiwan National University) and validated in previous studies (Moriguchi et al., 2005 (link); Yang et al., 2010 (link)). Monoclonal anti-total NKCC was from the Developmental Studies Hybridoma Bank (T4, Iowa City, IA). Antibody against α-tubulin (Cat #2125), rabbit anti-phospho AKT (Ser473; Cat# 9271), rabbit anti-AKT (Cat# 4691), rabbit anti-phospho ERK (Thr202/Tyr204; Cat# 4370), rabbit anti-ERK (Cat# 4695), and rabbit anti-phospho p70 S6k (T389; Cat# 9234) were from cell signaling (Beverly, MA). Mouse anti-p70 S6K (Cat# sc-8418) was purchased from Santa Cruz Biotechnology (Dallas, TX). BCA Protein Assay Kit (Cat #23227) was from Thermo Scientific (Rockford, IL). STS66 was synthesized by Töllner et al. (2014) (link) as described previously.