Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor

Rubén Martínez-Barricarte; Xiao-Fei Kong; Jean-Laurent Casanova

doi:10.21769/BioProtoc.3254

Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor

Bio Protoc. 2019 Jun 5;9(11):e3254. doi: 10.21769/BioProtoc.3254.

Authors

Rubén Martínez-Barricarte¹, Xiao-Fei Kong¹, Jean-Laurent Casanova^{1

2

3

4

5}

Affiliations

¹ St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York City, NY, USA.
² Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France.
³ Paris Descartes University, Imagine Institute, Paris, France.
⁴ Howard Hughes Medical Institute, New York, USA.
⁵ Pediatric Hematology and Immunology Unit, Necker Hospital for Sick Children, AP-HP, Paris, France.

Abstract

The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different subsets of PBMCs and immortalized B cells, in which SPPL2a is chemically inhibited and CD74-NTF levels are then assessed by flow cytometry or western blotting. The chemical inhibition of SPPL2a has been described elsewhere, but this is the first time that this inhibition has been reported as a protocol.

Keywords: 458; CD74-NTF; Chemical; Inhibition; L-685; SPPL2a.