*4.4. Antibodies*

Mouse anti-Kv4.2 (NeuroMab, Davis, CA, USA, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, rabbit anti-Kv4.2 (Sigma, St. Louis, MO, USA, P0233) was used at 1:2000 for western blot, pT602 (Santa Cruz, Dallas, TX, USA, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, Dallas, TX, USA, SC-22254-R) was used at 1:500 for western blot, and pp38 (Cell Signaling, Danvers, MA, USA, 4511s) at 1:1000 for western blot, 1:100 for immunostaining. Flag (Sigma, St. Louis, MO, USA, F3165) was used at 1:300 for immunostaining, actin (Sigma, St. Louis, MO, USA, A-1978) was used at 1:10,000 for western blot; Alexa Fluor 488 goa<sup>t</sup> anti-mouse (Invitrogen, Carlsbad, CA, USA, A-11029) was used at 1:500; Alexa Fluor 488 goa<sup>t</sup> anti-rabbit (Invitrogen, Carlsbad, CA, USA, A-11034) was used at 1:500; Alexa Fluor 555 goa<sup>t</sup> anti-mouse (Invitrogen, Carlsbad, CA, USA, A-21424) was used at 1:500; Alexa Fluor 555 goa<sup>t</sup> anti-rabbit (Invitrogen, Carlsbad, CA, USA, A-21429) was used at 1:500; Alexa Fluor 680 goa<sup>t</sup> anti-mouse (Invitrogen, Carlsbad, CA, USA, A-21057) was used at 1:10,000; Alexa Fluor 680 goa<sup>t</sup> anti-rabbit (Invitrogen, Carlsbad, CA, USA, A-21076) was used at 1:10,000; IRDye 800CW goa<sup>t</sup> anti-mouse (Licor, Licoln, NE, USA, 926-32210) was used at 1:10,000, IRDye 800CW goa<sup>t</sup> anti-rabbit (Licor, Licoln, NE, USA, 926-32211) was used at 1:10,000.

#### *4.5. Cell Culture and Transfection*

HEK-293T cells used in biochemistry experiments were obtained from Dr. Paul Worley's lab. HEK-293T cells were cultured in DMEM medium containing 10% FBS. Transfections were performed with X-tremeGENE 9 (Sigma, St. Louis, MO, USA, XTG9-RO) according to the manufacturer's specifications. Cells were harvested about 40 h after transfection.

#### *4.6. Western Blot and Quantification*

Protein samples were mixed with 4x LDS sample buffer (Invitrogen, Carlsbad, CA, USA, NP0007) and 10x sample reducing agen<sup>t</sup> (Invitrogen, Carlsbad, CA, USA, NP0007) to a final concentration of 1x. Samples were loaded on 4%–12% Bis-Tris gradient gel (Invitrogen, Carlsbad, CA, USA, 12-well, NP0322; 15-well, NP0323). The proteins were transferred to an Immobilon-FL PVDF membrane (EMD Millipore, Burlington, MA, USA, IPFL00010). The membrane was blocked with Odyssey blocking buffer (Li-COR, Licoln, NE, USA, 927-40000) for 1 h at room temperature, followed by incubation with primary antibody in PBS overnight at 4 ◦C. The membrane was then washed with PBST (PBS, pH 7.4 and 0.1% Tween-20) three times and incubated with secondary antibody in PBS for another hour. After three washes with PBS, the membrane was scanned using an Odyssey imaging system (LI-COR, Licoln, NE, USA) according to the manufacturer's protocol. Quantification of western blots was carried out using the gel analysis function in ImageJ within the linear range of detection, which is determined by using serial dilutions of a representative sample.
