Frap wizard software

FRAP studies were performed as described previously (26 (link)). Briefly, cells were seeded in eight-well imaging chambers (Ibidi, Martinsried, Germany) and transfected with the plasmid mCherry-Desmoglein1-N-18, a gift from Michael Davidson (Addgene plasmid # 55030; http://n2t.net/addgene:55030; RRID:Addgene_55030) using Turbofect (Thermo Fisher Scientific) according to the manufacturers protocol. 24 h after transfection cells were incubated with respective mediators and IgG fractions for 24 h. FRAP experiments were conducted on an SP5 inverted microscope with a x63 HC PL APO NA=1.2 objective (Leica, Wetzlar, Germany) in a cage incubator (OKOLAB, Burlingame, CA) at 37 °C at constant humidity with 5 % CO₂. The FRAP wizard software (Leica) was used to perform and analyze the experiments. Bleaching areas were chosen along the membrane of two transfected neighboring cells. 5 frames were captured to obtain the pre bleach mCherry intensity, followed by bleaching for 10 frames using the 594 nm laser line at 100 % transmission. Recovery of the fluorescence was recorded for 180 s until a stable fluorescence intensity was reached. The fraction of immobile molecules was calculated as
with I_plateau being the plateau fluorescence intensity after recovery and I_bleach being the fluorescence intensity after the bleaching step, both normalized to the initial fluorescence intensity.

Recovery curves were obtained by measuring the intensities of 18-μm² background, control and photobleached regions using Leica FRAP Wizard software. FRAP data acquisition and the parameters and equations used for the analysis are listed in the supplementary Materials and Methods and Table S6. GraphPad Prism software was used for nonlinear fitting and plotting of graphs.

For FRAP experiments wt and Pg-S665A murine keratinocytes were transiently transfected with pEGFP-C1-Dsg3 (kindly provided by Dr. Yasushi Hanakawa, Ehime University School of Medicine, Japan) using Lipofectamine 3000 (Invitrogen, Carlsbad, USA)^{82 (link),83} . Cells were switched to high Ca²⁺ (1.2 mM) 24 h after transfection and treated with vehicle or apremilast 48 h after Ca²⁺ switch for 2 h. FRAP experiments were conducted on an SP5 inverted microscope with a x63 HC PL APO NA = 1.2 objective (Leica, Wetzlar, Germany) in a cage incubator (OKOLAB, Burlingame, CA) at 37 °C at constant humidity with 5 % CO₂ using the FRAP wizard software (Leica). Region of Interest (ROI) for bleaching was chosen along the membrane of two transfected adjacent cells. Dsg3 signal of a ROI was bleached using an Argon laser ( $\lambda =488\mathrm{nm}$ ) at 100% transmission and recovery of the fluorescence was recorded for 180 s until a stable fluorescence intensity was reached. Immobile fraction and recovery halftime (τ_1/2) was determined to investigate Dsg3 mobility.