Xrs camera

Total cell and nuclear lysates were prepared as protocol using the following antibodies: STAT3, p-STAT3 (705), ERK, p-ERK, JNK, p-JNK, p-38, p-p38 followed by incubation with a horseradish peroxidase-conjugated secondary antibody and visualized using a Bio-Rad ChemiDoc XRS Imaging System with an XRS camera (Bio-Rad, Hercules, CA, U.S.A.).

AB8/13 cells, differentiated for 15 days at 37°C, treated with TNFα (10 ng/ml) for 4, 8 and 24 h were lysed by sonication in Radio-Immuno Precipitation Assay (RIPA) buffer (Sigma-Aldrich) for 1 min. Total protein concentration of the lysate was determined by BCA protein assay (Thermo Scientific) according to manufacturer’s protocol and equal amounts of lysate were mixed with SDS-loading buffer. The protein samples were electrophoresed on a SDS-(10%) PAA gel and transferred to a nitrocellulose membrane followed by blocking with 5% skimmed milk in PBS for 1 h. The membrane was cut based on the molecular masses of the target proteins followed by incubation with primary antibodies rabbit- polyclonal anti-VCAM-1 (~100-kDa, Santa Cruz Biotechnology Inc. Heidelberg, Germany), mouse-anti-human synaptopodin (~74-kDa, Progen), rabbit-polyclonal anti-GAPDH (~37-kDa, Santa Cruz Biotechnology Inc.) and/or mouse anti-human actin (~42-kDa, Millipore, Amsterdam, the Netherlands) for 1 h at room temperature. The membranes were washed three times with PBST and incubated with relevant horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Finally, the membranes were washed and the proteins on the membrane were visualized and detected using Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific) and a Gel Doc imaging system equipped with a XRS camera (Bio-Rad).

To analyse strains for the functional expression of a codon-optimised red-shifted luciferase, molten growth media were supplemented with d-luciferin potassium salt (Perkin Elmer) to give a final concentration of 0.3 mM. At indicated time points, plates were removed from the incubator and placed in the imaging chamber of a Bio-Rad gel-doc system equipped with a XRS+ camera. All filters were removed and the first picture was recorded under illumination to visualise the colonies present on the plate. A second picture was taken in the dark by opening the camera lens and using a picture acquisition time of 60 seconds. The pixel binning was kept at the highest resolution (254 dpi, lowest sensitivity) to keep the picture size identical to the illuminated picture. Pictures were exported to TIFF format.