Alexa fluor 488 fluorophore

To obtain quantitative information about the affinity of CSN5^ΔC to Nedd8, fluorescence anisotropy measurements were performed at 28°C, using a TECAN safire² plate reader (Tecan). The experiments were performed in 20 mM Hepes pH 7.5 and 75 mM NaCl. Alexa Fluor 488 fluorophore (Life Technologies) was used to label Nedd8 through a standard amine coupling procedure. The fluorescence was measured using excitation and emission wavelengths of 495 nm and 519 nm, respectively. CSN5^{ΔC,WT or.R106T} at varying concentrations (0–600 µM) was incubated in the presence of 4 nM Alexa Fluor 488-Nedd8. The same procedure was followed in the presence of CSN6^ΔC where a 1∶1 ratio of CSN5^ΔC/CSN6^ΔC was used to make the complex at the indicated concentrations. For each data point the measurement was done in triplicates and the recorded data corresponds to the average anisotropy. The data obtained were plotted and analysed using the IgorPro software. The Hill Equation was used for fitting the data in order to be able to deduct an estimate of the apparent equilibrium dissociation constant (K_D) of each complex between the protein or protein complex and Alexa Fluor 488-Nedd8. As there exists no evidence of cooperativity, in all cases, the data were fit with values of cooperativity equal to 1.

CIA-FLS were treated with 100 ng/ml recombinant IFN-γ (MilliporeSigma, Burlington, MA, USA) for 72 h in the presence or absence of 20 μM paxilline. To measure levels of MHC class II molecules, cells were scraped from culture dishes and left either intact or permeabilized with 0.5% saponin, followed by staining with an anti-MHC class II molecule antibody (clone HIS19; LSBio, Seattle, WA, USA), recognizing the RT1L haplotype expressed by Lewis rats [39 (link)], followed by a secondary antibody labeled with the Alexa Fluor 488 fluorophore (Life Technologies). For measurement of B7-H3, ICAM-1, and CD40 expression levels, cells were scraped from their culture flasks and stained with antibodies against B7-H3 (clone MIH42; BioLegend, San Diego, CA, USA) or CD40 (clone 5c3; BioLegend), followed by Alexa Fluor 488-conjugated secondary antibodies, or ICAM-1 (clone HA58; BD Biosciences, San Jose, CA, USA) conjugated to allophycocyanin (APC). Fluorescence was measured using a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

CHO cells transfected or not with pValac::dts::IL-4 and pValac::dts (negative control) were fixed with 2 % paraformaldehyde; were permeabilized with 1 % Triton X-100 (Sigma Aldrich); and were stained with an anti-IL-4 primary antibody [IL-4(NYRmIL-4) (Santa Cruz Biotechnology, Inc.)], a secondary antibody conjugated to the Alexa Fluor 488 fluorophore [Alexa Fluor 488 rabbit anti-rat IgG (H + L) conjugate (Life Technologies)], and 4′-6-diamidino-2-phenylindole (DAPI) (Life Technologies), as recommended by the manufacturers.
Images were captured with the Zeiss LSM510 META confocal microscope (Zeiss). The argon laser and filter set 09 were used with an emission wavelength greater than 510 nm to detect the Alexa Fluor 488 fluorophore (Life Technologies), while the epifluorescence and filter set 01 were used to detect DAPI (Life Technologies). All the images were visualized using LSM 5 Image Browser (Zeiss) software.