HEK293 cell lines were maintained in 1:1 DMEM F12/Opti-MEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified atmosphere with 5% CO2. Prior to transfection, cells were plated at a density of ~70%. Transient transfection of FERMT2 cDNA (cloned into a pcDNA4 vector; GeneArt) was performed using Fugene HD (Invitrogen) according to the manufacturer’s instructions. For WB, cells were washed with PBS and solubilized in ice-cold lysis buffer (Tris 1M pH 7.4; NaCl 1.5M; Nonidet P-40 0.1%; SDS 10%; sodium orthovanadate 100 mM; sodium deoxycholate 0.5%; 1x complete protease inhibitor mixture, Roche Applied Sciences). Cell extracts (5–20 μg) were analyzed using SDS-PAGE and the antibodies listed. For WB and immonufluorescence analysis, the following antibodies were used: hFERMT2 (GTX84507, GeneTex), amyloid precursor protein C-Terminal (A8717, Sigma), actin (A2066, Sigma), β-amyloid 6E10 (SIG-39320, Biolegends), ATPase (Na+-K+) alpha subunit (a5, DSHB), Rab4 (PA3-912, Thermo Scientific Pierce), Alzheimer precursor protein A4 clone 22C11 (MAB348, Millipore). For siRNA transfection, we used Dharmacon siRNA, non-targeting (D0018100105) and siFermt2 (J01275305, J01275306, J01276307, J01275308 and L01275300). Secreted Aβ and sAPP fragments were analyzed with an AlphaLISA, as described previously [15 (link)].
Sig 39320
Manufactured by BioLegend
Sourced in United States
SIG-39320 is a laboratory equipment product designed for use in scientific research. It serves as a core functional component without interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
A2066, by Merck Group (2 mentions)
A8717, by Merck Group (2 mentions)
Pcdna4 vector, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor mixture, by Roche (2 mentions)
Ab10148, by Abcam (1 mentions)
Amersham hybond p 0, by GE Healthcare (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Sig 39220, by BioLegend (1 mentions)
Goat anti rabbit igg hrp, by GeneTex (1 mentions)
Gtx213110 01, by GeneTex (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Gapdh antibody, by Proteintech (1 mentions)
Fluorescent secondary antibody, by Abcam (1 mentions)
Ab5076, by Abcam (1 mentions)
Mab348, by Merck Group (1 mentions)
A11005, by Thermo Fisher Scientific (1 mentions)
6 protocols using sig 39320
1
Modulation of Amyloid Precursor Protein in HEK293 Cells
2
Protein Immunoblotting for APP and Aβ
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Protein sample preparation and immunoblotting were performed according to our previous work [39 (link)]. The primary antibodies used for western blot were APP (1:1,000; SIG-39320, Biolegend, San Diego, CA) and anti-Aβ 1–42 (1:1,000; ab10148, Abcam, Cambridge, UK). Three independent experiments were performed.
3
Aβ Assembly Modulation by TDP-43 in APP/PS1ΔE9 Mice
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Western blot was performed to observe the changes in Aβ assembly after injecting TDP-43 to the APP/PS1ΔE9 mice. The concentration of the extracellular-enriched fraction and Triton-soluble fraction were determined and adjusted to the same protein concentration (48 μg in 20 μL). The samples were mixed with 6x loading buffer and heated in 95 °C for 10 min, then loaded into a 4–15% Tris-glycine gel and resolved at 100 V for 120 min. The separated proteins were transferred to a PVDF membrane (Amersham Hybond P 0.45, GE healthcare, Chicago, Illinois, USA) and blocked with 5% non-fat milk. The mouse monoclonal 6E10 antibody (1:5000, SIG-39320, BioLegend, San Diego, CA, USA) and mouse monoclonal 4G8 antibody (1:5000, SIG-39220, BioLegend, San Diego, CA, USA) were mixed and used for Aβ detection. A mouse monoclonal GAPDH antibody (1:5000, Proteintech, Rosemont, IL, USA) was used for loading control. The secondary antibody used for 6E10/4G8 mixture is goat anti-mouse IgG-HRP (GTX213111-01, Genetex, CA, USA) and for GAPDH is goat anti-rabbit IgG-HRP (GTX213110-01, Genetex, CA, USA). Immobilon Western Chemiluminescent HRP substrate (Merck, Darmstadt, Germany) was used for developing. The detection was carried out with Imagequant LAS4000 system (GE Healthcare, Life sciences, Hungary) and analyzed using ImageJ (NIH, Bethesda, MD, USA).
4
Immunohistochemical Analysis of Brain Tissue
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Immunohistochemical staining was performed on free-floating sections using either the Mouse or Rabbit Specific HRP/DAB IHC Detection Kit (ab236466, Abcam, United States) or fluorescent secondary antibodies (Abcam, United States). Unless otherwise noted, brain sections were blocked for 1 h with 5% normal serum of the species in which the secondary antibody was developed, followed by incubation with the primary antibody overnight at 4°C. Sections were then washed and incubated with secondary antibodies. Cresyl violet staining was performed according to the manufacturer’s instructions.
The following primary antibodies were used: rabbit anti-Iba1 (1:500; ab5076, Abcam, United States) and mouse anti-Aβ (1:1,000; SIG-39320, BioLegend, United States).
The following primary antibodies were used: rabbit anti-Iba1 (1:500; ab5076, Abcam, United States) and mouse anti-Aβ (1:1,000; SIG-39320, BioLegend, United States).
5
Transfection and Analysis of FERMT2 in HEK293 Cells
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HEK293 cell lines were maintained in 1:1 DMEM F12/Opti-MEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Prior to transfection, cells were plated at a density of ~70%. transient transfection of FERMT2 cDNA (cloned into a pcDNA4 vector; GeneArt) was performed using Fugene HD (Invitrogen) according to the manufacturer’s instructions. For WB, cells were washed with PBS and solubilized in ice-cold lysis buffer (Tris 1 M pH 7.4; NaCl 1.5 M; Nonidet P-40 0.1%; SDS 10%; sodium orthovanadate 100 mM; sodium deoxycholate 0.5%; 1× complete protease inhibitor mixture, Roche Applied Sciences). Cell extracts (5–20 μg) were analyzed using SDS-PAGE and the antibodies listed. For WB and immunofluorescence analysis, the following antibodies were used: hFERMT2 (GTX84507, GeneTex), amyloid precursor protein C-Terminal (A8717, Sigma), actin (A2066, Sigma), β-amyloid 6E10 (SIG-39320, Biolegends), ATPase (Na+–K+) alpha subunit (a5, DSHB), Rab4 (PA3-912, Thermo Scientific Pierce), Alzheimer precursor protein A4 clone 22C11 (MAB348, Millipore). For siRNA transfection, we used Dharmacon siRNA, non-targeting (D0018100105) and siFermt2 (J01275305, J01275306, J01276307, J01275308 and L01275300). Secreted Aβ and sAPP fragments were analyzed with an AlphaLISA, as described previously [15 (link)].
6
MERFISH Validation with Amyloid-beta Staining
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Once the MERFISH imaging process was completed, validation merslides were washed in 100% formamide for 15 min to remove fluorescent readout probes, rinsed with 1xPBS, and stained with 6E10 mouse anti-Aβ 1-16 monoclonal antibody (BioLegend #SIG-39320, 1:500 dilution, 2 h at RT) followed by PBS washes (5 min x3) and co-staining with 0.5% thioflavin S, and AlexFluor594-tagged goat anti-mouse IgG (ThermoFisher #A-11005, 1:600 dilution), and DAPI (10μg/ml) for 30 min at room temperature. Then the merslides were reassembled into a gasket chamber and immediately imaged using the Merscope Verification mode with the default settings for protein staining (DAPI, anti-mouse, and anti-rabbit channels "on", scan thickness: 10 μm).
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