Ab6709

The ChIP assay was performed as published12 (link). In brief, chromatin was harvested from T_FH/T_CM-like cells treated with and without IL-7 as indicated. Chromatin was incubated with antibodies to either STAT5 (Santa Cruz, sc-835x, 5 μg per IP) or IgG (Abcam, ab6709, 5 μg per IP) control and the precipitated DNA was analysed by qPCR with gene-specific primers (Supplementary Table 1). Samples were normalized to a standardized total input DNA control followed by subtraction of the IgG antibody as a control for the nonspecific background. The final value represents the percent enrichment of STAT5-specific signal.

The knee joints of mice with CIA were removed from the sacrificed mice for hematoxylin & eosin (H&E), Safranin O-fast green, and IHC analysis. Samples from each mouse were fixed in 4% buffered paraformaldehyde. Next, the tissues were cut into sections 3 μm in thickness, deparaffinized in xylene, and rehydrated by treating with a series of concentrations of ethanol. Tissue sections were prepared and stained with H&E or Safranin O-fast green. For IHC, heat-mediated antigen retrieval with sodium citrate buffer (pH6) was performed. After the inactivation of endogenous peroxidase, the sections were blocked by incubation in a 5% bovine serum album for 30 min at room temperature and then incubated overnight with rabbit anti-mouse IgG antibody (Abcam, ab6709, 1:1,000) at 4°C in a humidified chamber. After washing, the sections were incubated with HRP-conjugated anti-rabbit IgG (Abcam, ab6721, 1:1,000) for 1 h at room temperature. The reactions were developed using a DAB substrate kit. Each slide was evaluated by one of the authors under a microscope (Nikon, Tokyo, Japan). For histopathological evaluation, knee joint damage was scored under blinded conditions, using a widely used scoring system for the assessment of synovitis, cartilage degradation, and bone erosion (21 (link)).

CUT&Tag was performed with CUT&Tag-IT Assay Kit (53160, ACTIVE MOTIF) in 1.5 × 10⁶ FaDu cells using anti-SF3B2 (sc-514976, Santa Cruz, 5 µL, 1:20 dilution) and anti-H3 (ab1791, Abcam, 1 µL, 1:100 dilution) antibodies. Rabbit anti-mouse IgG (ab6709, Abcam, 1 µL) was used to enhance the signal. The cells were collected using a cell scraper.

The cell-permeable proteasome inhibitor MG132 (20 μM; C2211, sigma-adlrich, USA) was added to cellcultures for 6 h, followed by an immunoprecipitation lysis buffer containing protease and phosphatase inhibitors for 30 min. The lysates were immunoprecipitated with an anti-C1QBP antibody (ab101267, 1:500, Abcam, Cambridge, MA, USA) or immunoglobulin G (IgG) (ab6709, 1:500, Abcam, Cambridge, MA, USA) overnight at 4 °C on a rotating pattern. An antibody against ubiquitin (10201–2-AP, 1:1000 Proteintech, Rosemount, Minnesota, USA) was used to measure C1QBP ubiquitination. The immunoprecipitated proteins were quantified usingWB.

CoIP analysis was performed using the Pierce Co‐Immunoprecipitation Kit (#26149, Thermo Fisher Scientific) by following the manufacturer's instruction. In brief, antibodies of anti‐FUNDC1 (#ab224722, Abcam) and anti‐IgG (#ab6709, Abcam) were immobilized by amino link plus coupling resin. Cell lysates were precleared using the control agarose resin and then added to the spin column containing antibody‐coupled resin and incubated overnight at 4°C. The bound proteins were eluted by elution buffer, followed by immunoblotting analysis.

The cell cultures were treated with MG132 (10 M; C2211, Sigma-Adlrich, USA), a proteasome inhibitor that can penetrate cell membranes, for a duration of 6 hours. Following this, an immunoprecipitation lysis buffer containing phosphatase and protease inhibitors was introduced for an additional period of 30 minutes. Anti-PD-L1 antibody (ab101267, 1:500, Abcam, Cambridge, MA, USA) or IgG (ab6709, 1:500, Abcam, Cambridge, MA, USA) immunoprecipitated the lysates overnight at 4 ℃ in a rotating pattern. The assessment of PD-L1 ubiquitination was conducted using an antibody against ubiquitin (10201-2-AP, 1:1000 Proteintech, Rosemount, Minnesota, USA). The quantity of immunoprecipitated proteins was determined using western blotting.