Journal: PLOS One

Article Title: The neuronal chaperone proSAAS is highly expressed in the retina

doi: 10.1371/journal.pone.0321867

Figure Lengend Snippet: Panel A : ProSAAS processing diagram depicting paired basic proprotein convertase cleavage sites ( blue vertical lines ); epitopes for the three proSAAS antisera used in this study ( red, dark green, and light green horizontal lines ); and theoretical sizes of each proSAAS fragment derived from intact 27 kDa proSAAS by proprotein convertase cleavage. Panel B : Western blotting of mouse retinal and hippocampal ( hipp ) acid extracts using LS46 antiserum. Panel C: Western blotting of retinal lysates using proSAAS antibodies LS46 and 1E9. Each lane represents pooled retinal or hippocampal extracts taken from an individual animal. Panel D : Western blotting of a human retinal extract ( retina ) using LS46 antiserum.

Article Snippet: Primary antisera included the LS46 proSAAS antiserum described above; and antisera to choline acetyltransferase (ChAT; 1:500; Millipore; Burlington, MA; RRID: AB_94647), calbindin (1:500; Sigma-Aldrich; RRID: AB_476894); Brn3a (1:500; Synaptic Systems; Göttingen, Germany; RRID: AB_2737037), glial fibrillary acidic protein (GFAP; 1:500; Millipore; RRID: AB_211868); IIIβ-tubulin (1;500; Sigma-Aldrich; RRID: AB_1844090), RBPMS (RNA Binding Protein with Multiple Splicing; 1:500, GeneTex; Irvine, CA; RRID: AB_10720427); melanopsin antiserum (1:500; MilliporeSigma; Burlington, MA; Cat#: MABN770).

Techniques: Derivative Assay, Western Blot

Journal: PLOS One

Article Title: The neuronal chaperone proSAAS is highly expressed in the retina

doi: 10.1371/journal.pone.0321867

Figure Lengend Snippet: Panel A : ProSAAS-ir (LS46 staining) and RBPMS-ir co-staining showing proSAAS-ir surrounding RGCs. Panel B: Quantitative analysis of proSAAS-ir in each retinal layer, derived from 28 total images of retinal slices (n = 22 for LS46 antiserum-stained images; n = 6 for proSAAS ALS16157 antiserum-stained images; the choice of proSAAS antiserum was dictated by the available marker primary antibodies. Data are reported as corrected total fluorescence (CTF) normalized to the proSAAS-ir of the no-primary control in each experiment. Panel C : Stitched tilescan image of the optic nerve, optic nerve head, and surrounding retinal layers, depicting proSAAS LS46 and IIIβ-tubulin immunostaining. (* p < 0.05, *** p < 0.0001, **** p < 0.00001, unpaired t-test). (GCL = Ganglion Cell Layer, IPL = Inner Plexiform Layer, INL = Inner Nuclear layer, OPL = Outer Plexiform Layer, ONL = Outer Nuclear Layer, PR = Photoreceptor Layer).

Article Snippet: Primary antisera included the LS46 proSAAS antiserum described above; and antisera to choline acetyltransferase (ChAT; 1:500; Millipore; Burlington, MA; RRID: AB_94647), calbindin (1:500; Sigma-Aldrich; RRID: AB_476894); Brn3a (1:500; Synaptic Systems; Göttingen, Germany; RRID: AB_2737037), glial fibrillary acidic protein (GFAP; 1:500; Millipore; RRID: AB_211868); IIIβ-tubulin (1;500; Sigma-Aldrich; RRID: AB_1844090), RBPMS (RNA Binding Protein with Multiple Splicing; 1:500, GeneTex; Irvine, CA; RRID: AB_10720427); melanopsin antiserum (1:500; MilliporeSigma; Burlington, MA; Cat#: MABN770).

Techniques: Staining, Derivative Assay, Marker, Fluorescence, Control, Immunostaining

Journal: PLOS One

Article Title: The neuronal chaperone proSAAS is highly expressed in the retina

doi: 10.1371/journal.pone.0321867

Figure Lengend Snippet: Panel A: Quantitative image analysis of retinal cross sections labeled for proSAAS (LS46 antiserum) in cell-marker positive cells (GFAP, n = 4; calbindin, n = 3; CHaT, n = 6; RBPMS n = 4; Brn3a n = 4). Panel B : Quantitative image analysis of 6 retinal cross-sections, colabeled with proSAAS, Brn3a, and melanopsin antisera, showing no significant differences. Panel C : Representative image of proSAAS, melanopsin, and Brn3a staining. (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.0001, **** p < 0.00001, using one-way ANOVA with Tukey’s post-test for Panel A; and unpaired Student’s t-test for Panel B. ). For antiserum compatibility purposes, Abcepta ALS16157 goat anti-proSAAS antibody was used for the experiments in Panels B and C.

Article Snippet: Primary antisera included the LS46 proSAAS antiserum described above; and antisera to choline acetyltransferase (ChAT; 1:500; Millipore; Burlington, MA; RRID: AB_94647), calbindin (1:500; Sigma-Aldrich; RRID: AB_476894); Brn3a (1:500; Synaptic Systems; Göttingen, Germany; RRID: AB_2737037), glial fibrillary acidic protein (GFAP; 1:500; Millipore; RRID: AB_211868); IIIβ-tubulin (1;500; Sigma-Aldrich; RRID: AB_1844090), RBPMS (RNA Binding Protein with Multiple Splicing; 1:500, GeneTex; Irvine, CA; RRID: AB_10720427); melanopsin antiserum (1:500; MilliporeSigma; Burlington, MA; Cat#: MABN770).

Techniques: Labeling, Marker, Staining

Journal: PLOS One

Article Title: The neuronal chaperone proSAAS is highly expressed in the retina

doi: 10.1371/journal.pone.0321867

Figure Lengend Snippet: Panel A: ProSAAS and ChAT co-staining indicates that proSAAS is in both normal and displaced ChAT-positive amacrine cells, as well as in ChAT-positive synaptic layers in the IPL. Panel B : ProSAAS and GFAP co-staining depicts limited proSAAS staining in astrocytes. Panel C: ProSAAS and calbindin co-staining shows that proSAAS-ir is enriched in the cell bodies of calbindin-positive horizontal cells. Panel D: ProSAAS and Brn3a co-staining depicts proSAAS in and surrounding Brn3a-positive retinal ganglion cells. Panels A-D: All images represent maximum projection micrographs of fixed, cross-sectioned retina probed with proSAAS and cell-type marker antibodies. Brightness levels were manually adjusted for each experiment to increase contrast. LS46 proSAAS antiserum was used in this experiment.

Article Snippet: Primary antisera included the LS46 proSAAS antiserum described above; and antisera to choline acetyltransferase (ChAT; 1:500; Millipore; Burlington, MA; RRID: AB_94647), calbindin (1:500; Sigma-Aldrich; RRID: AB_476894); Brn3a (1:500; Synaptic Systems; Göttingen, Germany; RRID: AB_2737037), glial fibrillary acidic protein (GFAP; 1:500; Millipore; RRID: AB_211868); IIIβ-tubulin (1;500; Sigma-Aldrich; RRID: AB_1844090), RBPMS (RNA Binding Protein with Multiple Splicing; 1:500, GeneTex; Irvine, CA; RRID: AB_10720427); melanopsin antiserum (1:500; MilliporeSigma; Burlington, MA; Cat#: MABN770).

Techniques: Staining, Marker

Journal: PLOS One

Article Title: The neuronal chaperone proSAAS is highly expressed in the retina

doi: 10.1371/journal.pone.0321867

Figure Lengend Snippet: Log-transformed transcriptional counts of proSAAS ( Red ); clusterin (CLU, Blue ); and αcrystallin-β (CRYAB, Green ) transcripts in human and mouse studies obtained from ARCHS4, were normalized using the ComBat batch correction algorithm. Expression distributions are represented as a continuous density function, with vertical lines representing the means of each distribution. Panel A : Human: n = 2737 samples, 79 batches; Panel B: Mouse: n = 3045 samples, 155 batches).

Article Snippet: Primary antisera included the LS46 proSAAS antiserum described above; and antisera to choline acetyltransferase (ChAT; 1:500; Millipore; Burlington, MA; RRID: AB_94647), calbindin (1:500; Sigma-Aldrich; RRID: AB_476894); Brn3a (1:500; Synaptic Systems; Göttingen, Germany; RRID: AB_2737037), glial fibrillary acidic protein (GFAP; 1:500; Millipore; RRID: AB_211868); IIIβ-tubulin (1;500; Sigma-Aldrich; RRID: AB_1844090), RBPMS (RNA Binding Protein with Multiple Splicing; 1:500, GeneTex; Irvine, CA; RRID: AB_10720427); melanopsin antiserum (1:500; MilliporeSigma; Burlington, MA; Cat#: MABN770).

Techniques: Transformation Assay, Expressing

Journal: PLOS One

Article Title: The neuronal chaperone proSAAS is highly expressed in the retina

doi: 10.1371/journal.pone.0321867

Figure Lengend Snippet: Panels A, B : Differential cell expression plots. Panels B-C and E-F: UMAP projections and differential cell expression plots. Specific cell types are indicated by arrows in panels B and E , and by color in panels C and F . Relative proSAAS transcript expression is greatest in horizontal cells in both species, followed by RGCs. RGC subgroup expression is identifiable in the mouse data (seen as orange in panel F ). Amacrine cell interneurons are subdivided into different types in the human study ( panel A ), and as a single cell type in the mouse study ( panel D ). Panel G depicts a comparison of relative Pcsk1n mRNA expression in different RGC subtypes [data from [ , ]], while Panel H shows a comparison of changes in retinal PCSK1N expression in five disorders affecting the retina.

Article Snippet: Primary antisera included the LS46 proSAAS antiserum described above; and antisera to choline acetyltransferase (ChAT; 1:500; Millipore; Burlington, MA; RRID: AB_94647), calbindin (1:500; Sigma-Aldrich; RRID: AB_476894); Brn3a (1:500; Synaptic Systems; Göttingen, Germany; RRID: AB_2737037), glial fibrillary acidic protein (GFAP; 1:500; Millipore; RRID: AB_211868); IIIβ-tubulin (1;500; Sigma-Aldrich; RRID: AB_1844090), RBPMS (RNA Binding Protein with Multiple Splicing; 1:500, GeneTex; Irvine, CA; RRID: AB_10720427); melanopsin antiserum (1:500; MilliporeSigma; Burlington, MA; Cat#: MABN770).

Techniques: Expressing, Comparison