X63 hc pl apo na 1.2 objective

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.

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.