Active rac1 detection kit

Analysis of active Rac1 activity in the control cells and MAP7D3-depleted cells was carried out using Active Rac1 Detection kit (Cell Signaling Technology, CA, USA) according to the manufacturer’s instruction. Briefly, 100 µL of glutathione resin was transferred to a spin cup, and 20 µg of GST-PAK1-PBD was added. Next, 1 mg of total cell lysate was added to the spin cup and incubated at 4 °C for 1 h with gentle rocking. The spin cup was then subjected to centrifugation at 6,000 xg for 30 s. Following centrifugation, the spin cup was washed three times with 1X wash buffer at 6,000 xg for 30 s each. To elute the samples, the reducing sample buffer was added to the spin cup. Finally, the resulting samples were subjected to western blot analysis.

After serum starvation for 1 h, BHK-21 cells were infected with FMDV (MOI 10). After adsorption for 1 h at 4 °C, the cells were washed with cold PBS, shifted to 37 °C, and collected at indicated time points. Rac1 activation was then detected with the Active Rac1 Detection Kit (Kit #8815 Cell Signaling Technology, Inc.) in accordance with the manufacturer’s recommendations. In brief, GST-PAK1-PBD fusion protein was used to bind the activated form of GTP-bound Rac1, which can then be immunoprecipitated with glutathione resin. Rac1 activation levels were determined using Rac1 mouse mAb.

Rac1 activity was measured by pulldown assay using the active Rac1 detection kit (Cell Signaling), according to the manufacturer’s instructions. Cells were seeded in six-well plates (5 × 10⁵ cells per well) and maintained for 24 h in a CO₂ incubator. Cells were washed with cold PBS and lysed. Protein lysates were centrifuged and the supernatant was collected in new tubes containing beads pre-coupled with GST–PAK1-PBD and incubated under rotation at 4 °C for 60 min. The beads were washed and the proteins bound to them were separated by SDS-PAGE. The amounts of active Rac1 were determined by immunoblot analysis. Signals were detected using a LAS4010 imaging system (GE Healthcare, Chicago, IL, USA).

Rac1 activity assay was performed using the Active Rac1 Detection kit purchased from Cell Signaling Technology. Briefly, GST-PAK1-PBD fusion protein is used to bind the activated form of GTP-bound Rac1, which can then be immunoprecipitated with glutathione resin. Rac1 activation levels are then determined by western blot analysis using a Rac1 antibody.

Activation of both Rac1 and RhoA was measured by using the Active Rac1 Detection Kit (#8815, Cell Signaling, Danvers, MA, USA) and the Active Rho Detection Kit (#8820, Cell Signaling) respectively, according to the manufacturer’s instructions. In brief, single-cell ΔpBK-ITIH5 and mock clones were cultured in G418 containing growth medium for 48 h. Subsequent to the cell lysis, 550 μg of total cell protein lysate for each clone was mixed with 20 μg of GST-PAK1-PBD capturing (active) RAC1-GTP or GST-Rhotekin-RBD for RhoA. Glutathione matrix-immobilized Rac1-GTP or Rho-GTP was eluted in SDS sample buffer supplemented with DTT. After heat denaturation (5 min, 95 °C) Rac1 and RhoA proteins were detected by western blot analysis using specific antibodies (see Additional file 8). Total cellular RAC1 or RhoA protein was determined for each sample and used for normalization.