rabbit anti ap2a2  (Proteintech)

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Overview of work in the present study. The study began with an analysis of whole-exome sequencing (WES) data comparing rare genetic variant frequencies between late-onset Alzheimer’s disease (LOAD) cases and non-AD controls. Subsequent analyses focused on the MUC6 variable number of tandem repeat (VNTR) region. The particular variants identified initially to be associated with LOAD risk were later removed from the consensus variant calls, presumably because this VNTR region is extremely challenging for high-throughput sequence characterization methods. Polymorphism in the MUC6 VNTR region was associated with phospho-tau (pTau) pathology and with altered AP2A2 expression. Immunohistochemical analyses showed that AP2A2 protein was often colocalized with pTau tangles in LOAD brains. Green boxes indicate new data and analytic results.

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Sequencing, Variant Assay, High Throughput Screening Assay, Expressing, Immunohistochemical staining

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Plot of rare variant analysis in Alzheimer’s Disease Sequencing Project (ADSP) whole-exome sequencing (WES) data, indicating genetic variants associated with late-onset Alzheimer disease (LOAD) risk in the MUC6 variable number of tandem repeats (VNTR) region. (A) p Values are shown from single rare variant analyses in terms of association with LOAD phenotype. Several genetic variants were initially reported to be associated with LOAD risk at p < 1 × 10 −10 (red circles). These specific genetic variant calls later failed quality control when different data quality filters were applied. (B) Panel depicts this genomic region in schematic form. The initial genetic variants were present in a unique, exonic, and polymorphic VNTR region that is annotated inside the MUC6 gene and within 4000 bases of the 3′-untranslated region (UTR) of the AP2A2 gene. Whereas these specific SNP calls in the MUC6 VNTR region were the initial reason for studying this genetic locus, the subsequent experiments focused on the polymorphic size of the VNTR region and the association with pTau proteinopathy.

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Variant Assay, Sequencing, Control

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Comparison of AP2A2 transcript levels (a proxy for gene expression) from cerebellum of 2 groups (n = 15 in each group) stratifying on the size of largest MUC6 VNTR region. The 2 groups were matched for age at death, sex, severity of Alzheimer disease neuropathologic changes, and postmortem interval ( Table 3 ). Fold-change of detected AP2A2 transcript relative to a different transcript was calculated based on quantitative PCR results. Regardless of the normalization method, there was a trend for the subjects with larger VNTR regions (red squares) to have lower average levels of detected AP2A2 transcripts in comparison to the individuals with smaller VNTR regions (blue circles). This difference was statistically significant when AP2B1 levels were used for normalization (A) , but was only a nonsignificant trend when GAPDH (B) was used for normalization. Comparisons were performed using unpaired 2-tailed Student t -tests.

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Comparison, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Brightfield immunohistochemical staining of AP2A2 in human brain sections. Shown are representative results from staining of temporal neocortex. Panels (A) and (B) show results from a nondemented aged subject with minimal Alzheimer-type pathology. Panel (A) and ( B ; higher magnification) show the relatively even, neuronal staining for AP2A2. Panels (C) and (D) show representative results from a demented subject with severe Alzheimer’s disease pathology. Note that the staining pattern of AP2A2 is here reminiscent of neurofibrillary tangles (green arrowhead), as well as some scattered, darkly AP2A2-immunoreactive structures in the neuropil (black arrows). Scale bars: A , C = 200 microns; B , D = 25 microns. Panel (E) shows the results of Western blots using the same AP2A2 antiserum—immunoblots of HEK-293 cell extracts are shown. This antiserum recognizes a prominent band at the predicted (∼104 kDa) size that is augmented by transfection with a pCMV-AP2A2 plasmid. The human brain extracts (shown here are Western blots from the cerebellum of 1 control and 1 LOAD brain) stained a similar banding pattern.

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Immunohistochemical staining, Staining, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: AP2A2 and pTau colocalization, detected using immunofluorescence and digitally quantified in a convenience sample of 5 LOAD cases. (A) A representative example of AP2A2 and pTau (immunostained with PHF1 antibody) staining using epifluorescence microscopy, as well as the HALO software generated digital markup showing the area of staining that was AP2A2 + , pTau + , and AP2A2 + pTau + double positive. In the colocalized markup, the red indicates pTau + , the green indicates AP2A2 + , and the yellow indicates AP2A2 + pTau + double positive. (B) and (C) Confocal z-stack images show cells double positive for pTau and AP2A2 at higher magnification. The arrow and arrowhead indicate the same cells (B) , shown in the orthogonal projection images in (C) . (D) Panel shows the percentage of the tissue in each of the regions of interest that was positive staining. The number of AP2A2 + pTau + double positive cells in relation to the number of AP2A2 + is shown in (E) , and in relation to the pTau + cells in (F) , as quantified by confocal microscopy in the hippocampus and neocortex. Data are plotted as mean ± SD for the 5–10 z-stacks included for each region and for each slide.

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Immunofluorescence, Staining, Epifluorescence Microscopy, Software, Generated, Confocal Microscopy

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Unlike in cases with LOAD pathology, AP2A2 does not colocalize extensively with phospho-TDP-43 in brains with limbic-predominant age-related TDP-43 proteinopathy neuropathologic changes (LATE-NC), nor with pTau in progressive supranuclear palsy (PSP) brains. (A) A representative example of AP2A2 and phospho-TDP-43 staining using epifluorescence microscopy shows the lack of colocalization of AP2A2 with phospho-TDP-43 in a brain with both AD neuropathologic changes (ADNC) and LATE-NC. (B) AP2A2 was also not colocalized with pTau (immunostained with antibody PHF1) in the caudate nucleus of subjects with autopsy-proven PSP. Quantitative analyses of these results are shown in and .

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Staining, Epifluorescence Microscopy

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Western blots show results of experiments in cultured cells testing whether AP2A2 and Tau proteins can be coimmunoprecipitated. Flag-AP2A2 and Tau expressing plasmids were transfected into cultured HeLa cells, separately and together. The lysates, Anti-Flag M2 coimmunoprecipitation (Co-IP), and nonimmunized mouse serum (NMS) controls were immunoblotted for AP2A2 (A) , Tau (B) , AP2B1 (C) , and tubulin as a control (D) . Molecular weights (kDa) are indicated on the left of the blots. A band labeled by the AP2A2 antibody (∼104 kDa) was present without transfection, and that signal was augmented in the lysate and in IPs where Flag-AP2A2 plasmids were transfected, indicating that Flag-AP2A2 transfection was successful. Anti-Flag M2 beads pulled down the Flag-tagged AP2A2. There were no augmented signals of Tau protein in the M2-IP product of the AP2A2 and Tau cotransfection when probed with Tau antibody DA9, indicating that AP2A2 and Tau proteins were not coimmunoprecipitated. Bands at ∼55 kDa (near to Tau) in the NMS and M2-IP lanes were likely immunoglobulin protein and were present in all the immunoblots. As a positive control, endogenous AP2B1, a known binding partner of AP2A2 with the same molecular weight of ∼104 kDa, was Co-IP’d with AP2A2. Gel portions where the proteins were predicted to be present, according to their known molecular weights, are shown with a red asterisk for each protein. Complete Western blots are shown in .

Article Snippet: For AP2A2 immunohistochemistry, rabbit anti-AP2A2 (Cat no. LC-C482433/126430, LSBio, Seattle, WA, 1:1000 dilution) was used, and manufacturer’s recommendations were followed for light microscopic immunohistochemistry.

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Co-Immunoprecipitation Assay, Control, Labeling, Cotransfection, Positive Control, Binding Assay, Molecular Weight

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Overview of work in the present study. The study began with an analysis of whole-exome sequencing (WES) data comparing rare genetic variant frequencies between late-onset Alzheimer’s disease (LOAD) cases and non-AD controls. Subsequent analyses focused on the MUC6 variable number of tandem repeat (VNTR) region. The particular variants identified initially to be associated with LOAD risk were later removed from the consensus variant calls, presumably because this VNTR region is extremely challenging for high-throughput sequence characterization methods. Polymorphism in the MUC6 VNTR region was associated with phospho-tau (pTau) pathology and with altered AP2A2 expression. Immunohistochemical analyses showed that AP2A2 protein was often colocalized with pTau tangles in LOAD brains. Green boxes indicate new data and analytic results.

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Sequencing, Variant Assay, High Throughput Screening Assay, Expressing, Immunohistochemical staining

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Plot of rare variant analysis in Alzheimer’s Disease Sequencing Project (ADSP) whole-exome sequencing (WES) data, indicating genetic variants associated with late-onset Alzheimer disease (LOAD) risk in the MUC6 variable number of tandem repeats (VNTR) region. (A) p Values are shown from single rare variant analyses in terms of association with LOAD phenotype. Several genetic variants were initially reported to be associated with LOAD risk at p < 1 × 10 −10 (red circles). These specific genetic variant calls later failed quality control when different data quality filters were applied. (B) Panel depicts this genomic region in schematic form. The initial genetic variants were present in a unique, exonic, and polymorphic VNTR region that is annotated inside the MUC6 gene and within 4000 bases of the 3′-untranslated region (UTR) of the AP2A2 gene. Whereas these specific SNP calls in the MUC6 VNTR region were the initial reason for studying this genetic locus, the subsequent experiments focused on the polymorphic size of the VNTR region and the association with pTau proteinopathy.

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Variant Assay, Sequencing

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Comparison of AP2A2 transcript levels (a proxy for gene expression) from cerebellum of 2 groups (n = 15 in each group) stratifying on the size of largest MUC6 VNTR region. The 2 groups were matched for age at death, sex, severity of Alzheimer disease neuropathologic changes, and postmortem interval ( Table 3 ). Fold-change of detected AP2A2 transcript relative to a different transcript was calculated based on quantitative PCR results. Regardless of the normalization method, there was a trend for the subjects with larger VNTR regions (red squares) to have lower average levels of detected AP2A2 transcripts in comparison to the individuals with smaller VNTR regions (blue circles). This difference was statistically significant when AP2B1 levels were used for normalization (A) , but was only a nonsignificant trend when GAPDH (B) was used for normalization. Comparisons were performed using unpaired 2-tailed Student t -tests.

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Brightfield immunohistochemical staining of AP2A2 in human brain sections. Shown are representative results from staining of temporal neocortex. Panels (A) and (B) show results from a nondemented aged subject with minimal Alzheimer-type pathology. Panel (A) and ( B ; higher magnification) show the relatively even, neuronal staining for AP2A2. Panels (C) and (D) show representative results from a demented subject with severe Alzheimer’s disease pathology. Note that the staining pattern of AP2A2 is here reminiscent of neurofibrillary tangles (green arrowhead), as well as some scattered, darkly AP2A2-immunoreactive structures in the neuropil (black arrows). Scale bars: A , C = 200 microns; B , D = 25 microns. Panel (E) shows the results of Western blots using the same AP2A2 antiserum—immunoblots of HEK-293 cell extracts are shown. This antiserum recognizes a prominent band at the predicted (∼104 kDa) size that is augmented by transfection with a pCMV-AP2A2 plasmid. The human brain extracts (shown here are Western blots from the cerebellum of 1 control and 1 LOAD brain) stained a similar banding pattern.

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Immunohistochemical staining, Staining, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: AP2A2 and pTau colocalization, detected using immunofluorescence and digitally quantified in a convenience sample of 5 LOAD cases. (A) A representative example of AP2A2 and pTau (immunostained with PHF1 antibody) staining using epifluorescence microscopy, as well as the HALO software generated digital markup showing the area of staining that was AP2A2 + , pTau + , and AP2A2 + pTau + double positive. In the colocalized markup, the red indicates pTau + , the green indicates AP2A2 + , and the yellow indicates AP2A2 + pTau + double positive. (B) and (C) Confocal z-stack images show cells double positive for pTau and AP2A2 at higher magnification. The arrow and arrowhead indicate the same cells (B) , shown in the orthogonal projection images in (C) . (D) Panel shows the percentage of the tissue in each of the regions of interest that was positive staining. The number of AP2A2 + pTau + double positive cells in relation to the number of AP2A2 + is shown in (E) , and in relation to the pTau + cells in (F) , as quantified by confocal microscopy in the hippocampus and neocortex. Data are plotted as mean ± SD for the 5–10 z-stacks included for each region and for each slide.

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Immunofluorescence, Staining, Epifluorescence Microscopy, Software, Generated, Confocal Microscopy

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Unlike in cases with LOAD pathology, AP2A2 does not colocalize extensively with phospho-TDP-43 in brains with limbic-predominant age-related TDP-43 proteinopathy neuropathologic changes (LATE-NC), nor with pTau in progressive supranuclear palsy (PSP) brains. (A) A representative example of AP2A2 and phospho-TDP-43 staining using epifluorescence microscopy shows the lack of colocalization of AP2A2 with phospho-TDP-43 in a brain with both AD neuropathologic changes (ADNC) and LATE-NC. (B) AP2A2 was also not colocalized with pTau (immunostained with antibody PHF1) in the caudate nucleus of subjects with autopsy-proven PSP. Quantitative analyses of these results are shown in and .

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Staining, Epifluorescence Microscopy

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Western blots show results of experiments in cultured cells testing whether AP2A2 and Tau proteins can be coimmunoprecipitated. Flag-AP2A2 and Tau expressing plasmids were transfected into cultured HeLa cells, separately and together. The lysates, Anti-Flag M2 coimmunoprecipitation (Co-IP), and nonimmunized mouse serum (NMS) controls were immunoblotted for AP2A2 (A) , Tau (B) , AP2B1 (C) , and tubulin as a control (D) . Molecular weights (kDa) are indicated on the left of the blots. A band labeled by the AP2A2 antibody (∼104 kDa) was present without transfection, and that signal was augmented in the lysate and in IPs where Flag-AP2A2 plasmids were transfected, indicating that Flag-AP2A2 transfection was successful. Anti-Flag M2 beads pulled down the Flag-tagged AP2A2. There were no augmented signals of Tau protein in the M2-IP product of the AP2A2 and Tau cotransfection when probed with Tau antibody DA9, indicating that AP2A2 and Tau proteins were not coimmunoprecipitated. Bands at ∼55 kDa (near to Tau) in the NMS and M2-IP lanes were likely immunoglobulin protein and were present in all the immunoblots. As a positive control, endogenous AP2B1, a known binding partner of AP2A2 with the same molecular weight of ∼104 kDa, was Co-IP’d with AP2A2. Gel portions where the proteins were predicted to be present, according to their known molecular weights, are shown with a red asterisk for each protein. Complete Western blots are shown in .

Article Snippet: Sections were blocked in 5% normal goat serum in TRIS-buffered saline (5% S+TBS) for 1 hour at room temperature, then incubated in both primary antibodies anti-AP2A2 (rabbit polyclonal, 1:100 dilution; Cat no. LC-C482433/126430, Lifespan Biosciences) and PHF1 (mouse monoclonal, 1:500 dilution, gift from Dr Peter Davies), diluted in 5% S+TBS, for 22 hours at 4 °C.

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Co-Immunoprecipitation Assay, Labeling, Cotransfection, Positive Control, Binding Assay, Molecular Weight