Protein a g conjugated agarose beads

Jurkat-sh-Ctr or Jurkat-sh-NLRX1 cells (5 ×10⁷) were washed twice by PBS and then resuspended in 1 ml lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail, phosphatase inhibitor cocktail, pH 7.4). Cell lysates were rotated at 4°C for 30 min to obtain complete cell lysis, and cell debris was pelleted by centrifugation at 16,000 × g for 30 min at 4°C. Partially cleared lysates (80 µl) were kept for use as the input for immunoblotting. Immunoprecipitation was performed by incubating cell lysates with 10 µg anti-NLRX1 or control mouse IgG1 antibody (5415, Cell Signaling) plus 50 μl protein A/G conjugated agarose beads (53133, ThermoFisher Scientific) for 8 hours, followed by 3 times wash with lysis buffer. The proteins pulled down by antibodies were subjected to immunoblotting with antibodies specific for NLRX1, FASTKD5, PRDX3, and SPNS1. To determine the interaction of NLRX1 and FASTKD5 upon the HIV-1 infection, 1 ×10⁸ Jurkat cells were infected with 100 ng p24 HIV-1 R3A. At 72 hpi, immunoprecipitation and immunoblotting were conducted as described above.

The spleens of the indicated mice were collected, minced and filtered through 100 μm cell strainer to get single cells. The red blood cells were removed using the ACK buffer (Gibco). Then the splenocyte lysates were prepared in the lysis buffer (1% Triton X-100, 150 mM NaCl, 3 mM MnCl₂, 1 mM CaCl₂, 1 mM MgCl₂, 25 mM Tris-HCl, pH 7.6) with protease inhibitor cocktail (Sigma-Aldrich). For immunoprecipitation, cell lysates were pre-cleared with Protein A/G-conjugated agarose beads (Thermo Fisher Scientific) at 4°C for 2 hours with rotation, then incubated with corresponding antibodies or control IgG overnight at 4°C. The cell lysates were then incubated with Protein A/G beads for an additional 2 hours. The beads were washed four times with lysis buffer and re-suspended in SDS sample buffer for western blot analysis.
For immunoprecipitation of CD24Fc receptors, the spleen cells were incubated with control IgGFc, CD24Fc or desialylated CD24Fc (10 μg/ml) for 1 hour. After washing away the unbound CD24Fc with PBS, the spleen cells were lysed in lysis buffer. Protein A/G beads were used to pull down Fc. The amounts of Siglec-E associated with CD24Fc were determined by immunoblotting.