Ab65986

Protein was obtained after extracting RNA from the samples with Trizol reagent (Invitrogen). Protein was extracted from the same sample, precipitated and washed accordingly to the manufactures protocol. The protein was solubilized in lysis buffer with 1% sodium dodecyl sulfate. β-mercaptoethanol was added as a reducing agent making up 5% of the solution. Protein concentration was determined in triplicates on NanoDrop Spectrophotometer (NanoDrop 2000c, Thermo Scientific, Waltham, MA). The proteins were separated using 4–10% bis–tris gel (Invitrogen) under gel electrophoresis and transferred to a polyvinylidene difluoride membrane using an iblot machine (Invitrogen). The membrane was blocked and incubated in primary antibody overnight diluted 1:1000 for Aqp1 (ab168387, Abcam, Cambridge, UK), Lamin-B1 (ab65986, Abcam) and Nkcc1 (14581, Cell Signaling Technology, Danvers, MA). Membranes were incubated in secondary antibody diluted 1:10,000 and the protein bands were detected using chemiluminescent (Amersham ECL, GE Healthcare, Chicago, IL). The reaction was captured on luminescent image analyzer (LAS-4000 Luminescent Image Analyzer, Fujifilm, Tokyo, Japan). Protein band was quantification using ImageJ (NIH, USA). The relative protein expression is related to the loading control.

The expression levels of GPR120, IL-6, IL-8, and SOX9 were assessed via western blot analysis. Briefly, proteins were extracted from ATDC5 chondrocytes using cell lysis buffer (Cell Signaling Technologies, USA) supplemented with protease inhibitor cocktail. Following centrifugation at 10000 × g, the supernatant protein was collected from the samples. A BCA protein concentration assay was used to assess the protein concentrations in accordance with the manufacturer’s manual (Sigma-Aldrich, USA). Then, 20 μg protein was loaded onto an 8-12% SDS-PAGE gel for electrophoresis and transferred onto a PVDF membrane (Bio-Rad, USA). To block the nonspecific binding sites, the samples were incubated with 5% non-fat milk in TBST, followed by primary antibodies overnight at 4 °C, and HRP-conjugated secondary antibody at room temperature (RT) for 1 h. The protein bands were then visualized via ECL chemiluminescence (Thermo Scientific, USA). The resulting optical density was quantified using Image J software (NIH, USA). The following antibodies were used in this study: GPR120 (1:1000, #ab230869, Abcam, USA); type II collagen (1: 500, #MAB8887, Chemicon, USA); aggrecan (1: 500, #ab3778, Abcam, USA); SOX-9 (1:1000, #ab59252, Abcam, USA); β-actin (1: 10000, #ab8227, Abcam, USA); NF-κB p65 (1:1000, #ab16502, Abcam, USA); Lamin B1 (1: 5000, ab65986, Abcam, USA).

Western blot analysis of equal amounts of crosslinked chromatin extracts or of histone extracts (see Supplemetary Methods) was performed as described^{25 (link)}. Antibodies used for western blot analysis are the following: anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07-449, Millipore), 8WG16 (05-952, Millipore), anti-elongating RNA pol II (ab5095, Abcam), anti-Lamin B1 (ab65986, Abcam), anti-histone 4 (ab10158, Abcam), and anti-histone3 (ab1791, Abcam). Dilutions of antibodies were performed according to the manufacturer's guidelines. Time for analysis is indicated in the figures (Fig. 4b; Supplementary Fig. 4a, b; Supplementary Fig. 3c, d). Uncropped scans of all western blot figures are supplied in the Source Data file.

Podocytes were lysed and homogenized in RIPA buffer for 30 min on ice, then centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was aspirated and added to 5 × protein loading buffer. The proteins were separated by SDS-PAGE gel electrophoresis and transferred to on a polyvinylidene fluoride membrane (Millipore). Membranes were blocked with 5% nonfat dry milk in TBST buffer for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C. Blots were incubated with the corresponding primary antibodies. The primary antibodies used are as follows: APC antibody (ab40778, Abcam), PLA2R1 antibody (NBP1-84449, Novus Biologicals), β-Actin antibody (4967, Cell Signaling), p-ERK1/2 antibody (9101, Cell Signaling), ERK1/2 antibody (4695, Cell Signaling), YB-1 antibody (4202, Cell Signaling), Lamin B1 antibody (ab65986, Abcam). The next day was incubated with the corresponding secondary antibody. ECL exposure solution was used to visualize Blots (Thermo Fisher Scientific).

RNA IP (RIP) for HuR protein was performed using RNA-Binding Protein Immunoprecipitation Kit (Cat.#17-700, Millipore) according to the manufacturer's instructions. Cell lysates were prepared with RIP lysis buffer and then incubated with anti-HuR antibodyor normal mouse IgG (ab65986, Abcam) at 4°C for 6 h with rotation. RNA-protein complexes were retrieved by magnetic beads (Magnetic Beads Protein A/G, CS203178), washed 6 times in the cold RIP wash buffer. After proteinase K treatment, beads were separated using a magnetic separator from the sample. The supernatant was directly resuspended in TRIzol reagent and subjected to RNA extraction. Then, qRT-PCR analysis was performed, and the RNA levels in RIP samples were normalized to input samples.

After protein content determination with Bradford reagent, 50 µg of total protein or100 µg of nuclear protein was boiled in Laemmli sample buffer and submitted to 8% SDS‐PAGE gels. Proteins were transferred to Inmunoblot nitrocellulose membrane (BioRad Inc) and, after blocking with 5% non‐fat dry milk, incubated overnight with different antibodies as indicated: anti‐HIF1α (1:1000, 10006421, Cayman Chemical, Hamburg, Germany), anti‐HIF2α (1:1000, ab199, Abcam, Cambridge, UK) and anti‐CD36 (1:1000, NB400‐144, Novus, Abingdon, UK). Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) Western blotting protocol (BioRad Inc). The anti‐βactin (1:5000, A‐5441, Sigma‐Aldrich Inc) and the anti‐lamin B (1:1000, ab65986, Abcam) antibodies were used as loading control for total and nuclear protein respectively. Densitometric analysis of the bands was performed using Image J software (NIH, Bethesda, MD).