Mouse igg1 mopc 21

The anti-EV-A71 mAbs MA28-7 (mouse IgG₁) [11 (link)] and MA105 (mouse IgG_2b) [23 (link)], were generated from mice immunized with EV-A71-1095. The anti-EV-A71 mAb 10F0 (IgG₁) was purchased from Abcam. Anti-DAF antibody (IF7, mouse IgG_2b) [34 (link)] was used to inhibit Coxsackievirus B3 binding to HeLa cells. For negative controls, mouse IgG₁ (MOPC-21) and IgG_2b (MOPC-141) were purchased from Biolegend and Sigma-Aldrich, respectively. Soluble recombinant forms of human proteins fused to the Fc region of human IgG₁ (PSGL-1-Fc, SCARB2-Fc, and CTLA-4-Fc) were purchased from R&D Systems. CTLA-4-Fc was used as a negative control Fc protein.

Monoclonal antibodies (mAbs) to human integrins were purchased as follows: β1 (TS2/16) (Biogems, Westlake Village, CA), β3 (VIPL2) (Abcam), β5 (AST-3T) (Biolegend), α5 (NKI-SAM-1) (Biolegend), and αV (P2W7) (LSBio, Seattle, WA, USA). Anti-mouse integrin MAbs were also obtained: β1 (HMb1-1) (Biolegend), β3 (HMb3-1) (Biolegend), β5 (KN52; Thermo Fisher Scientific), α5 (5H10-27) (Biolegend), and αV (RMV-7) (Biolegend). Isotype-matched control antibodies were also obtained: mouse IgG1 (MOPC-21, Biolegend), mouse IgG2a (MOPC-173) (Biolegend), mouse IgG2b (MPC-11) (Biolegend), Armenian hamster IgG (HTK888) (Biolegend), and rat IgG1 (RTK2071) (Biolegend). The cells were stained with those fluorescently labeled antibodies, washed twice with PBS containing 2% FBS and 2 mM EDTA (Wako, Osaka, Japan), and analyzed by using a BD Accuri C6 flow cytometer and software (BD Biosciences, San Jose, CA, USA).

HIS-tagged recombinant human Growth and Differentiation Factor-15 was produced in HEK293T cells (Invigate GmbH, Jena). The anti-human GDF-15 antibody used in this manuscript was generated in female C57BL/6J ^GDF-15-/–mice kindly provided by Dr. Jens Strelau (Heidelberg, Germany) and validated for binding affinity by surface plasmon resonance at NMI (Reutlingen, Germany). Specificity was tested by tissue cross-reactivity studies. Antibody for cell culture and in vivo experiments was produced by Exbio (Prague, Czech Republic). For experiments in humanized mice, a humanized version of this antibody was generated by grafting the complementarity determining regions on a hinge-stabilized human IgG4 backbone, followed by recombinant expression (Evitria, Schlieren, Switzerland). For experiments in fully immune-competent mice, a surrogate antibody (0297.mIgG1) was also produced by Evitria. To block GFRAL, clone 3P10, described in Suriben, R. et al. Antibody-mediated inhibition of GDF15-GFRAL activity reverses cancer cachexia in mice. Nat Med 26, 1264-1270 (2020), and in US20170306031A1 was used. Isotype antibodies were anti-Fluorescein [4-4-20 (enhanced)] Human IgG4 S228P (Absolute antibodies) or mouse IgG1 MOPC-21 (Biolegend). Unless otherwise indicated, antibodies were used according to the manufacturer´s instructions.