Rabbit anti human nfκb p65

Frozen tissue sections were fixed for 15 min with 4% paraformaldehyde (Immunofix, Bio-Optica, Milan, Italy; 05-K01015) in PBS, washed twice in PBS and permeabilized for 10 min with 0.5% Triton X-100 (Merck; X100) in PBS. After 1 h block with 1% bovine serum albumin (BSA, Merck; A3294) at room temperature, coverslips were incubated in a humidified chamber for 2 h at room temperature with a mouse anti-human HIF-1α (H1 alpha 67), (Novus Biologicals, LLC, Littleton CO, USA), rabbit anti-human NFκB p65, (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human VEGFA ab46154, (Abcam, Cambridge, UK). Afterward, coverslips were washed with PBS 3 times (5 min/wash) and incubated for 1 h with goat anti-rabbit or goat anti-mouse IgG Alexa Fluor 555 fluorescent secondary antibody (1:200, Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 100nM SYTOX™ green (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA). Finally, samples were washed with PBS 3 times (5 min/wash) and coverslips were mounted in ProLong Diamond Antifade Mountant (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA) and analyzed with an LSM510 confocal microscopy (Zeiss, Oberkochen, Germany). Quantification of the fluorescence intensity in the tubules and stroma of the control and FD patient was determined using Image J Software v1.51 (NIH, Bethesda, MD, USA).

Cells were fixed with 37% formaldehyde, lysed by RIPA buffer, and sonicated. After preclearing with agarose beads, the cell lysate was incubated with 5 μg rabbit anti-human NF-κB p65 (Santa Cruz) or isotype IgG (Santa Cruz). The IgG was gathered by sperm DNA-treated agarose beads, followed by treatment with RNase A (Takara) and proteinase K (Roche Applied Science, Mannheim, Germany). The TNFA DNA promoter that coprecipitated with p65 was quantified by real-time PCR using a forward primer (5’-AGTCAGTGGCCCAGAAGACC-3’) and a reverse primer (5’-GGCGGGGAAAGAATCATTCAACC-3’).

Cells were seeded in 96-well plates at 100% confluence and cultured for 2 days. Apyrase treatment (10 U/mL) was added to some wells for 10 min prior to scratching. The monolayer of cells was scratched using a sterile pipette tip 10, 15, and 30 min (CREB phosphorylation) or 15, 30, and 60 min (NFκB activation) before fixation. Some wells were treated with 0.6 μM of rMASP-1 after scratching. Cells were fixed in ice-cold methanol–acetone (1:1) for 10 min. Cells were stained with rabbit anti-human phospho-CREB (1:200 Cell Signaling Technology Inc., Danvers, MA, USA) antibody or rabbit anti-human NFκB p65 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) antibody followed by Alexa568 conjugated goat anti-rabbit IgG (1:500) and Hoechst 33342 (1:50,000, Invitrogen, Waltham, MA, USA) (Table 1). Photographs were taken using an Olympus IX-81 fluorescence microscope. All analyses were performed using the original unmodified images using CellP software (version 5.2). Nuclear mean red fluorescence (pCREB) or the ratio of the cytoplasmic and nuclear mean red fluorescence (NFκB) was calculated.