ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition. 