Neon electroporation pipette tips

For each electroporation reaction, 8 × 10⁵ NIH 3T3 Cav1-knock in cells^{66 (link)} suspended in 10.5 µl of resuspension buffer R were mixed with the indicated amount of antibody mixture diluted in 2 µL of PBS. mRNAs were added immediately prior to electroporation, to limit the degradation by potential RNAse activity. Cav1-GFP mRNA encoding Vhh-Fc (WT or PRYSPRY binding-deficient H433A mutant) and TRIM21 were electroporated. The cell mRNA mixtures were taken up into 10 µL Neon electroporation pipette tips (Invitrogen) and electroporated using the following settings: 1400 V, 20 ms, 2 pulses (as described in refs. ^{18 (link),67 (link)}). Electroporated cells were transferred to antibiotic-free Fluorobright media supplemented with 10% FBS and left to incubate for 5 h in an incubator before the cells were harvested for immunoblotting. GFP fluorescence measured using an Incucyte® (essenbioscience) and was normalized to the control (Vhh-Fc^H433A). Protein detection was performed using the following antibodies: Fc: goat anti-hIgG Fc broad 5211–8004 (1:2000); TRIM21: rabbit anti-TRIM21 D101D (ST#9204) (1:1000), Vinculin: rabbit anti-Vinculin EPR8185 ab 217171 (1:50,000); and Caveolin-1: rabbit anti-Cav1 (BD: 610059, 1:1000).

Anti-IKK alpha antibody (ab169743, Abcam) was delivered into cells using the Neon Transfection system (Invitrogen). For each electroporation reaction, 8 × 10⁵ cells suspended in 10.5 µl of Resuspension Buffer R were mixed with the indicated amount of antibody diluted in 2 µl of PBS. The cell antibody mixtures were taken up into 10 µl Neon electroporation pipette tips (Invitrogen) and electroporated using the following settings: 1400 V, 20 ms, 2 pulses (as described in ref. ^{7 (link)}). Electroporated cells were transferred to antibiotics-free DMEM supplemented with 10% FBS and left to incubate for 3–5 h in an incubator before the cells were harvested for immunoblotting. Cells were primed with human Interferon alpha (PeproTech) at 24 h before electroporation with a final concentration of 1000 U ml⁻¹.