Active rho detection kit

After the cell culture medium was removed, cells were washed twice with ice-cold PBS. The procedures were conducted according to the recommendations of Active Rho Detection Kit (Kit # 8820, Cell Signaling, USA). In this method, the GST-Rhotekin-RBD fusion protein binds to the active form of RhoA (RhoA-GTP) and then immunoprecipitated with glutathione, whereas the inactive form of RhoA (RhoA-GDP) is removed during washing steps. RhoA-GTP expression was detected by western blotting using an anti-RhoA antibody.

The level of activated RhoA GTPases in cells after RF-EMF exposure was determined with an active Rho detection kit purchased from Cell Signaling Technology according to previous work (Sin et al., 2020 (link)). Cells were collected and lysed in 1× lysis buffer containing 1 mM PMSF. Followed this, the lysates were harvested by centrifuge at 16,000 × g at 4°C for 15 min. The supernatants were incubated with glutathione-S-transferase (GST) agarose beads coupled to Rhothekin RBD recombinant protein, which is used to bind the activated form of GTP-bound RhoA. Then, the GTP-bound RhoA was immunoprecipitated with glutathione resin. Activated RhoA GTPases pull-downs were released by boiling for 5 min in a 2× SDS Sample Buffer with 200 mM dithiothreitol. Bound RhoA was detected by western blot with rabbit anti-RhoA (1:1000, CST, United States).

Rho activation was measured using Active Rho detection kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, HMEC‐1 cells were seeded in 75 cm² flask to grow to a 70%‐80% confluence and treated with 1 μM IMB5046 for 30 minutes, then exposed to 10 μM PF‐228 or not for 10 minutes. Subsequently, cells were lysed and incubated with GST‐Rhotekin‐RBD and glutathione resin to selectively isolate and pull down the active form of Rho. The active form was detected by immunoblotting using a Rho antibody (8789, Cell Signaling Technology). Densitometric values of immunoblots were obtained using ImageJ. The values were corrected by the density of total Rho and normalized to the control expressed as 1. The experiment was repeated three times.

The cells were lysed on ice after being rinsed twice with ice‐cold PBS. Then, the lysate protein concentrations in the lysates were determined using a BCA protein assay. According to the instructions of the Active Rho Detection Kit (Cell Signaling Technology, USA), the GST‐Rhotekin‐RBD fusion protein was used to bind the activated Rho protein. The levels of the activated form of RhoA, GTP‐RhoA, were determined by western blotting using a RhoA rabbit antibody (Proteintech, USA).

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.

The detection of active RhoA was conducted according to the protocol of Active Rho Detection Kit purchased from Cell Signaling Technology. Briefly, the hippocampus tissue was isolated and lysated with 500 μL 1× Lysis Buffer(add 1 mM PMSF before using). BCA method was used to measure the concentration of protein. First, the Glutathione Resin suspension was centrifuged to discard the liquid, then 400 μg GST‐Rhotekin‐RBD was added before the sample was added to the spin cup. After shaked at 4°C for 1 hour, sample was washed with the Lysis Buffer twice and transferred to a new collecting cup, 50 μL 2× reducing sample buffer (200 mM DTT before use) was added to the spin cup and vortexed, incubated at room temperature for 2 minutes, centrifuged at 4°C. Finally, the liquid was collected, boiled for 5 minutes, and analyzed by western blot.

Immunoblotting was performed as described previously. The sources of the primary antibodies were as follows: anti-RACGAP1 (Proteintech, 1:1,000), anti-DICER1 (Proteintech, 1:1,000), and anti-β-actin (Proteintech, 1:1,000), p-ERK1/2 (CST, 1:1,000), total ERK (CST, 1:1,000), Active Rho Detection kit (#8820), anti-mouse and anti-rabbit peroxidase conjugated secondary antibodies were purchased from Cell Signalling Technology (Danvers, MA).