Expression and localization of GPR91 and GPR99 in murine organs
Cell and Tissue Research
Julia Diehl, Barbara Gries, Uwe Pfeil, Anna Goldenberg, Petra Mermer, Wolfgang Kummer,
Renate Paddenberg
Table S1: Primary antibodies
Antibody
CatalogID;
company
rabbit antiLS-A3315;
human GPR91 MBL, LifeSpan
antibody
BioSciences
rabbit antiLS-A1865;
human GPR99 LifeSpan
antibody
BioSciences
Information on the data sheets and comments
On the data sheets the following is given: The antibody
was raised against a synthetic KLH conjugated 15 amino
acid peptide from the 2nd cytoplasmic domain of human
GPR91 and LifeSpan BioSciences predicts reactivity with
murine GPR91 since BLAST analysis revealed 93%
identity with mouse.
Comment: We run a BLAST analysis using UniProt
(http://www.uniprot.org/uniprot/P16284 ) to clarify if
other murine proteins exhibit similarities to the amino acid
sequence 125 to 137 representing the 2nd cytoplasmic loop
of human GPR91 but scored only murine GPR91. Novus
Biologicals has offered a rabbit anti-human GPR91
antibody under the designation NLS3315 which was
raised against a synthetic KLH conjugated peptide made
to the 2nd cytoplasmic loop and the company specified that
the peptide showed 93% identity and 93% homology to
mouse. Since in the data sheets of MBL and Novus
Biologicals - and also of RayBiotech; Code 119-10076 one and the same picture of anti-GPR91 stained human
kidney medulla was shown we assume that the antibodies
sold as LS-A3315 and NLS3315, respectively, were
identical and we used the peptide NLS3315PEP from
Novus Biologicals for preabsorption controls.
On the data sheet the following information is given: The
antibody was raised against a synthetic 17 amino acid
peptide from the 2nd extracellular domain of human
OXGR1. LifeSpan BioSciences predicts reactivity with
murine GPR99 since BLAST analysis revealed 94% identity
with mouse.
Comment: We made enquiries using UniProt
(http://www.uniprot.org/uniprot/P16284) to find that the 2nd
extracellular loop is built up by 29 amino acids in the
positions 173 to 201. When we performed a BLAST analysis
for mouse just one hit was obtained: GPR99 with 89.7%
identity. When we performed the analysis with the first 17
amino acids of this loop, 17 amino acids in the middle or at
the end of the loop we got 94.1%, 88.2%, and 88.2% identity
mouse
monoclonal
anti-human VATPase B1/2
(F-6) antibody
sc-5544; Santa
Cruz
Biotechnology
sheep anti-rat
renin antibody
AP00945PU-N;
Acris
monoclonal
anti-Actin, αSmooth
Muscle-FITC,
antibody
produced in
mouse; clone
1A4
F3777; SigmaAldrich
so that we assume that a peptide corresponding to the initial
17 amino acids to the loop was used for immunization. In
our BLAST analysis no hit with murine proteins other than
GPR99 was obtained. Based on these data combined with
our preabsorption controls using blocking peptide LS-P1865
(LifeSpan BioSciences) we assumed that the
immunolabeling with this antibody was specific.
On the data sheet the following information is given: The
antibody was raised against amino acids 334-513 mapping at
the C-terminus of V-ATPase B1 of human origin. On the
data sheet a western blot is shown in which V-ATPase B1/2
expression was analysed in mouse kidney extract. The
antibody reacted with a single protein of the expected size of
56 kDa.
Comment: Gao et al. (Gao X, Eladari D, Leviel F, Tew BY,
Miró-Julià C, Cheema FH, Miller L, Nelson R, Paunescu
TG, McKee M, Brown D, Al-Awqati Q (2010) Deletion of
hensin/DMBT1 blocks conversion of β- to α-intercalated
cells and induces distal renal tubular acidosis. Proceedings
of the National Academy of Sciences of the United States of
America 107(50):21872–21877) have used this antibody for
immunolabeling of the B1 subunit of the V-ATPase in
murine kidneys.
On the data sheet the following information is given: The
antibody was raised against recombinant rat prorenin and
reacts with renin of rat and mouse.
On the data sheet the following information is given:
Immunogen: N-terminal synthetic decapeptide of α-smooth
muscle actin. The antibody is specific for the single isoform
of α-smooth muscle actin. It reacts specifically with αsmooth muscle actin in immunoblotting assays and labels
smooth muscle cells in frozen or formalin-fixed, paraffinembedded tissue sections. Species reactivity: rabbit, mouse,
guinea pig, chicken, goat, sheep, snake, human, frog, rat,
canine, bovine.
Figure legend
Table S1: Information on primary antibodies provided by the companies and our comments
on them.