B cell supernatants containing secreted antibodies were diluted in
H2O, 4x sample loading buffer (Life Technologies) and 10x sample reducing agent
(Life Technologies) and loaded onto precast gels with a 4-12% acrylamide gradient
(Invitrogen). The iBlot2 apparatus (Life Technologies) was used for protein transfer to
PVDF membranes followed by blocking for 1 h at room temperature with 3% BSA in TBS. The
membrane was incubated with different combinations of primary and secondary antibodies
diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to
wash the membrane between incubations. For detection of IgG, anti-human IgG-biotinylated
antibody (Southern Biotech, 2040-08) was used at 1 µg ml-1, followed by
25 ng ml-1 streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch,
016-030-084). IgM isotypes were stained with 10 µg ml-1 unlabeled goat
anti-human IgM (Southern Biotech, 2020-01) and 8 ng ml-1 donkey anti-goat HRP
(Jackson ImmunoResearch, 705-036-147). To detect LAIR1-containing antibodies, a polyclonal
goat anti-human LAIR1 antibody (R&D) at 2 µg ml-1 was combined
with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a
Las4000 imager (General Electric Company).
H2O, 4x sample loading buffer (Life Technologies) and 10x sample reducing agent
(Life Technologies) and loaded onto precast gels with a 4-12% acrylamide gradient
(Invitrogen). The iBlot2 apparatus (Life Technologies) was used for protein transfer to
PVDF membranes followed by blocking for 1 h at room temperature with 3% BSA in TBS. The
membrane was incubated with different combinations of primary and secondary antibodies
diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to
wash the membrane between incubations. For detection of IgG, anti-human IgG-biotinylated
antibody (Southern Biotech, 2040-08) was used at 1 µg ml-1, followed by
25 ng ml-1 streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch,
016-030-084). IgM isotypes were stained with 10 µg ml-1 unlabeled goat
anti-human IgM (Southern Biotech, 2020-01) and 8 ng ml-1 donkey anti-goat HRP
(Jackson ImmunoResearch, 705-036-147). To detect LAIR1-containing antibodies, a polyclonal
goat anti-human LAIR1 antibody (R&D) at 2 µg ml-1 was combined
with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a
Las4000 imager (General Electric Company).