Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: The cells were stained with APC-CD11b antibody (Miltenyi 130-113-793) and CD45 antibody (Miltenyi 130-118-687) for 30 min with three subsequent washes.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 1. One-hybrid identification of LPP as a PEA3 interaction partner. (A) Diagrammatic representation of full-length LPP and the truncated version isolated in a yeast one-hybrid assay. The locations of the LIM domains and the Gal4 fused transcriptional activation domain (AD) are shown. (B) Yeast one-hybrid interactions between PEA3 and LPP(254–612) on an ETS site-driven -galactosidase reporter gene. The presence of the empty expression plasmids pMSe4 and pGAD10, pMSe4 containing zebra fish PEA3, or pGAD10 containing LPP(254– 612) is indicated.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: Isolation, Y1H Assay, Activation Assay, Expressing

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 2. LPP expression in normal and cancerous breast tissue. (A) Immunohistochemical analysis of LPP protein expression in normal human breast tissue sections. An enlarged part of the image is shown to illustrate expression in the ductal epithelial cells. (B) LPP expression from human tissue derived from patients with invasive ductal carcinomas. LPP expression is revealed by brown staining. A control IgG-stained adjacent section is shown in the bottom panel. (C) Immunofluorescence analysis of LPP expression (green, fluorescein isothiocyanate) and PEA3 expression (red, tetramethyl rhodamine isothiocyanate) in ductal carcinoma samples. (D) Confocal analysis of LPP expression (fluorescein isothiocyanate; green) in MDA-MB-231 cells. Nuclei are visualized with DAPI staining.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: Expressing, Immunohistochemical staining, Derivative Assay, Staining, Control

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 3. LPP is required for PEA3 target gene activity. (A to C) Reverse transcription-PCR analysis of endogenous MMP-1 expression. (A) 293 cells were transfected with the indicated combinations of expression vectors for PEA3 (400 ng), MEK(N-S218E/S222D) (200 ng) and LPP (400 ng). (B) 293 cells were transfected with expression vectors for PEA3 (400 ng), MEK(N-S218E/S222D) (200 ng) and the indicated LPP or control siRNAs. (C) MDA-MB-231 cells were trans- fected with the indicated LPP or control siRNAs.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: Activity Assay, Reverse Transcription, Expressing, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 4. LPP potentiates the transactivation activity of PEA3. Lucifer- ase reporter gene assays using a PEA3 site-driven (A to C) or a COX-2 promoter-driven luciferase reporter (D) in 293 cells. Data are presented relative to the activity of the reporter alone (taken as 1). Western blots showing the expression levels of GAPDH or PEA3 in the presence and absence of LPP are shown below the graphs. (A) LPP alone (2 g) or mouse PEA3 (200 ng) and increasing amounts of LPP (0, 0.5, 1, and 2 g) were cotransfected. (B) Mouse PEA3 (200 ng), constitutively active MEK, and increasing amounts of LPP (0, 1, and 2 g) were cotransfected. , addition of 2 g of LPP. (C) Mouse PEA3 (200 ng) and increasing amounts of LPP (0, 1, and 2 g) were cotransfected. , addition of 2 g of LPP. Cells were either serum starved or stimulated with PMA in the presence or absence of the MEK inhibitor U0126 where indicated. (D) Mouse PEA3 (200 ng), constitutively active MEK, and increasing amounts of LPP (0, 1, and 2 g) were cotransfected.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: Activity Assay, Luciferase, Western Blot, Expressing

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 5. LPP is required for PEA3-mediated promoter activation. Luciferase reporter gene assays were carried out in the presence of the indicated LPP or control siRNAs with the PEA3 site-driven (600 ng; A, B, and D) or Cox-2 promoter-driven (600 ng; C) luciferase reporter constructs. 293 cells were transfected with PEA3 expression constructs (600 ng) in the absence (A and C) and presence (B) of PMA stimu- lation. (D) MDA-MB-231 cells were transfected with reporter alone. Western blots showing the expression levels of LPP, PEA3, and GAPDH in the presence and absence of the indicated RNAi con- structs are shown below the graphs.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: Activation Assay, Luciferase, Control, Construct, Transfection, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 6. Mapping the PEA3 binding surface(s) on LPP. (A) GST pull-downs using GST or GST-PEA3 proteins and in vitro translated LPP derivatives. A diagrammatic representation of full-length LPP is shown at the top, and truncated derivatives of this are depicted next to appropriate lanes. (B) GST pull-downs of GST or GST-LPP(254–612) and in vitro translated zebra fish PEA3, human ER81, and human ERM. Ten percent input protein is shown. (C) Coimmunoprecipitation of LPP and PEA3. 293 cells were transfected with a PEA3 expression vector and immuno- precipitations (IP) were carried out with control IgG or LPP antibodies. Immunoprecipitated LPP and PEA3 were detected by immunoblotting (IB). Inputs show equal amounts of LPP and PEA3. (D) Coimmunoprecipitation of endogenous LPP and ER81 from MDA-MB-231 cells. Immunoprecipitations (IPs) were carried using anti-LPP antibody (top panel) or anti-ER81 antibody (bottom panel), and precipitated LPP and ER81 were detected by immunoblotting (IB) with the appropriate antibodies. (E and F) Reporter gene analysis of the PEA3 site-driven luciferase reporter construct in 293 cells. (E) Mouse PEA3 (200 ng) and the indicated Gal4-LPP constructs (2 g) were cotransfected. (F) Mouse PEA3, human ER81, or human ERM (200 ng) and constitutively active MEK and LPP (2 g) were cotransfected. The increase in activation (n-fold) by LPP is indicated above each set of bars.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: Binding Assay, In Vitro, Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Luciferase, Construct, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: The LIM Domain Protein LPP Is a Coactivator for the ETS Domain Transcription Factor PEA3

doi: 10.1128/mcb.01667-05

Figure Lengend Snippet: FIG. 7. LPP is recruited to PEA3-regulated promoters in vivo. (A) ChIP assay of endogenous LPP on a PEA3 site-driven luciferase reporter in 293 cells. The presence of transfected PEA3 is indicated. Immunoprecipitations (IP) were carried out with either LPP antibody, PEA3 antibody, rabbit IgG (R), or mouse IgG (M) as controls. Co- precipitating DNA was revealed by PCR with promoter (prom) or coding region (cod)-specific primers as indicated in the schematic. Input DNA was diluted 10-fold before amplification. (B) ChIP assay of endogenous LPP on the MMP-1 promoter in MDA-MB-231 cells. Immunoprecipitations(IPs) were carried out with antibodies against either LPP, PEA3, or IgG as a control (con). Coprecipitating DNA was revealed by PCR with MMP-1 promoter or GAPDH coding region- specific primers.

Article Snippet: Cells grown in 100-mm-diameter dishes were cross-linked with 1% formaldehyde for 5 min. Immunoprecipitations were carried out using the antibodies anti-PEA3 (Santa Cruz), anti-LPP (MP2 [29]), or nonspecific IgG (Upstate).

Techniques: In Vivo, Luciferase, Transfection, Control