Protein a agarose bead

Cells were incubated with MG132 (5 μM) for 4 h and cell extracts were prepared in buffer containing complete protease inhibitor (Roche) and deubiquitinase inhibitor N-Ethylmaleimide (E3876; Sigma Aldrich). The lysates were incubated with IRF-1 or anti-HA antibody overnight at 4 °C, and protein complexes were pelleted with protein A-agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) and separated by SDS/PAGE. Ubiquitinated IRF-1 was immunoblotted with anti-HA or anti-IRF-1 antibody.

The CB-enriched fraction (FPEL; 0.5 mg) isolated from the testes of WT and GRTH KI mice was reverse cross-linked and pre-cleared using 50 μl of protein A-agarose beads (Thermo Scientific) and 1 μg of rabbit IgG containing IP binding buffer (Thermo Scientific, United States) for 30 min at 4°C in a rocker. Upon spinning, the resulting pre-cleared supernatant obtained was incubated with 5 μg of anti-GRTH rabbit polyclonal antibody (Kavarthapu et al., 2019 (link)) or rabbit IgG at 4°C overnight to co-immunoprecipitate the GRTH–RNP complex. Upon overnight incubation, the GRTH–RNP complex was incubated with 50 μl of protein A-agarose beads and incubated for 2 h at 4°C. The resulting protein–RNP complex was washed with IP binding buffer (four washes), and the total RNA was isolated using the phenol/chloroform/isoamyl alcohol (25:24:1, v/v; Thermo Scientific, Waltham, MA, United States) method. The first-strand cDNA was prepared using iscript first-strand synthesis kit (Bio-Rad Laboratories, Hercules, CA, United States), and qRT-PCR was performed with Fast SYBR green using a set of gene-specific primers (Supplementary Table 1) in a 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA, United States).

Immunoprecipitation was conducted using a 100 µg of total proteins, employing an IP-grade Integrin α11 antibody or IgG (1 mg/mL) (Sigma-Aldrich), incubated for 16 h at 4 °C. Subsequently, Protein A agarose beads (50 µL) (Thermo Scientific) were added in the immunoprecipitated solution and allowed to incubate for 4 h at 4 °C. The resultant immune complexes were collected through centrifugation at 3000× g for 2 min at 4 °C. The collected pellet was washed with PBS and then re-suspended in 25 µL of 1× sample buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 0.002% Bromophenol Blue, 0.7135 M (5%) β-mercaptoethanol, 10% glycerol), followed by heating at 95 °C for 5 min. After centrifugation at 12,000× g for 30 s at 4 °C, supernatant containing the IP samples was collected.

Single-cell PCR analysis was applied in the case of seven healthy controls and eight HIV-infected individuals (Table S2 in Supplementary Material). The 96-well PCR plate containing sorted cells was incubated at 65°C for 4 h to reverse the crosslinking. RT-PCR was performed using the QIAGEN OneStep RT-PCR kit following the manufacturer’s instructions. Two rounds of PCR reactions were used to amplify IgH, Igκ, or Igλ genes from single cells as previously described (24 (link)). To facilitate subcloning, a third round of PCR was performed. Third-round PCR products with unique restriction enzyme digestion sites were subcloned into corresponding Igγ1, Igκ, or Igλ expression vectors. The resulting plasmids were sequenced, and paired IgH and IgL genes from the same well were cotransfected into 293T cells [American Type Culture Collection (ATCC) CRL-3216] to express recombinant mAbs. Culture supernatant was collected 7 days after transfection and Abs were purified using Protein A Agarose beads (ThermoFisher Scientific, Madison, WI, USA).