lrp1 cluster ii protein  (MedChemExpress)

Journal: Journal of Cancer

Article Title: Anti-PAI-1 Monoclonal Antibody Inhibits the Metastasis and Growth of Esophageal Squamous Cell Carcinoma

doi: 10.7150/jca.77888

Figure Lengend Snippet: PAI-1 mAb inhibited migration in an LRP1-dependent manner. A. Interaction between PAI-1 and LRP1 detected by co-immunoprecipitation. B. qRT-PCR (left) and Western blot (right) analysis of LRP1 knockdown in KYSE30lm3 cells. C. Knockdown of LRP1 counteracted PAI-1 protein-promoted migration. D. Knockdown of LRP1abolished mAb-1E2 and 2E3-inhibited migration. E. mAb-2E3 inhibited the phosphorylation level of STAT1 and failed to inhibit the level of metastasis-related target proteins which were detected in cells under the treatment of antibody. F. Western blot of LRP1, pSTAT1 and total STAT1 in KYSE30lm3 cells transfected with siRNAs against LRP1. GAPDH was used as a loading control. G. Grayscale analysis of pSTAT1 and total STAT1 levels in KYSE30lm3 cells transfected with siRNAs against LRP1. H. qRT-PCR of STAT1 transcripts in KYSE30lm3 cells transfected with siRNAs against LRP1. * P< 0.05, ** P< 0.01, *** P< 0.001, ns ≥0.05.

Article Snippet: Recombinant Human LRP1-Cluster II-Fc Chimera protein (2368-L2) was purchased from Bio-Techne.

Techniques: Migration, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Transfection

Journal: Journal of Cancer

Article Title: Anti-PAI-1 Monoclonal Antibody Inhibits the Metastasis and Growth of Esophageal Squamous Cell Carcinoma

doi: 10.7150/jca.77888

Figure Lengend Snippet: MAb-2E3 blocking the binding of LRP1-Cluster II-Fc and PAI-1. A. The binding activity of PAI-1 and LRP1-Cluster II-Fc protein was determined and EC50 value was calculated. B. mAb-2E3 was able to block the binding between LRP1-Cluster II-Fc and PAI-1. mIgG was used as the control. IC50 values were calculated.

Article Snippet: Recombinant Human LRP1-Cluster II-Fc Chimera protein (2368-L2) was purchased from Bio-Techne.

Techniques: Blocking Assay, Binding Assay, Activity Assay

Journal: Communications biology

Article Title: LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin.

doi: 10.1038/s42003-022-03873-0

Figure Lengend Snippet: Fig. 3 LRP1- and Megalin-mediated Tcnα uptake require cell-surface sGAG. a TcsL, Tcnα, TcsH, TcdB, and TpeL were incubated with Heparin-Sepharose at 4 °C for 1 h. The protein samples were prepared from the input, toxin-bound heparin beads, and the supernatant was separated on an SDS-PAGE and detected by coomassie blue staining. b The HeLa WT or SLC35B2‒/‒ cells were transfected with mock, Ldlr, LRP1CII-LdlrC, and MegalinCII-LdlrC. The cells were then incubated with 200 nM Tcnα on ice for 30 min, changed with the fresh medium, and incubated at 37 °C for 3 h. Representative images are shown. Red fluorescence (mCherry) marked transfected cells. The scale bar represents 50 μm. c The round-shaped cells among all mCherry-positive cells shown in b were quantified and plotted in a bar chart. Error bars (n = 6) indicate mean ± SD, n.s. not significant, two-sided Student’s t-test.

Article Snippet: The following antibodies, reagents, and recombinant proteins were purchased from the indicated vendors: Alexa Fluor 488 goat anti-rabbit IgG (ab150077, 1:1000, Abcam), rabbit polyclonal IgG against β-Actin (ab227387, 1:5000, Abcam), rabbit monoclonal IgG against LDLR (ab52818 for western blot, 1:500; ab30532 for immunofluorescence, 1:200; Abcam), rabbit monoclonal IgG against LRP1 (ab92544, 1:20000 for western blot and 1:200 for immunofluorescence, Abcam), HRP-conjugated goat anti-human IgG-Fc antibody (SSA001, 1:3000, Sino Biological), Hoechst 33258 staining solution (E607301, BBI), NHS-Rhodamine fluorescent labeling kit (#46406, Thermo Fisher Scientific), recombinant human LRP1 Cluster II Fc chimera (R&D Systems, 2368-L2), Precast PAGE Gel (abs9309, Absin), Polyethylenimine Linear (PEI) MW25000 (40816ES03, YEASEN), and Heparin-Sepharose (Abcam, ab193268).

Techniques: Incubation, SDS Page, Staining, Transfection

Journal: Communications biology

Article Title: LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin.

doi: 10.1038/s42003-022-03873-0

Figure Lengend Snippet: Fig. 4 LDLR versus LRP1 in various cells. a The mRNA levels of LDLR, LRP1, and Megalin in MCF-7, THP-1, PC-3, A549, HeLa, HepG2, BJ, U-87 MG, and Caco-2 cells are shown. Data were obtained from a public database (http://www.proteinatlas.org). b The protein levels of LDLR and LRP1 in the HeLa, MCF, MEF, BJ, and U-87 MG cells were measured by immunoblot analysis. c The depletion of LDLR and LRP1 in the U-87 MG LDLR‒/‒ and LRP1‒/‒ cells showed by immunoblot analysis. Actin served as a loading control. The experiments in b, c have been repeated independently twice with similar results. d Immunofluorescence analysis shows that Alexa Fluor 555-labeled Tcnα (50 nM) is robustly bound to the U-87 MG WT, LDLR−/−, and LRP1−/−cells. Cell nuclei were stained with Hoechst dye. Representative images are shown. The scale bar represents 50 μm. e Immunofluorescent staining shows cellular localization of LDLR and endocytosed Tcnα in the U-87 MG WT, LDLR−/−, and LRP1−/−cells. Cell nuclei were stained with Hoechst. Representative images are shown. Scale bars represent 10 μm. f Colocalization of LDLR and endocytosed Tcnα in the U-87 MG WT, LDLR−/−, and LRP1−/−cells were analyzed by software ImageJ ver1.53. The percentage of the Tcnα signals that overlapped with LDLR in each cell was calculated and plotted as an open circle. Error bars (n = 10) indicate mean ± SD, ***P < 0.001 versus WT, two-sided Student’s t-test.

Article Snippet: The following antibodies, reagents, and recombinant proteins were purchased from the indicated vendors: Alexa Fluor 488 goat anti-rabbit IgG (ab150077, 1:1000, Abcam), rabbit polyclonal IgG against β-Actin (ab227387, 1:5000, Abcam), rabbit monoclonal IgG against LDLR (ab52818 for western blot, 1:500; ab30532 for immunofluorescence, 1:200; Abcam), rabbit monoclonal IgG against LRP1 (ab92544, 1:20000 for western blot and 1:200 for immunofluorescence, Abcam), HRP-conjugated goat anti-human IgG-Fc antibody (SSA001, 1:3000, Sino Biological), Hoechst 33258 staining solution (E607301, BBI), NHS-Rhodamine fluorescent labeling kit (#46406, Thermo Fisher Scientific), recombinant human LRP1 Cluster II Fc chimera (R&D Systems, 2368-L2), Precast PAGE Gel (abs9309, Absin), Polyethylenimine Linear (PEI) MW25000 (40816ES03, YEASEN), and Heparin-Sepharose (Abcam, ab193268).

Techniques: Western Blot, Control, Labeling, Staining, Software

Journal: Communications biology

Article Title: LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin.

doi: 10.1038/s42003-022-03873-0

Figure Lengend Snippet: Fig. 5 The U-87 MG LDLR−/−and LRP1−/−cells are more resistant to Tcnα. a U-87 MG WT, LDLR−/−, and LRP1−/−cells were incubated with 30 pM Tcnα at 37 °C for 20 h. Cytopathic effect was observed in the U-87 MG WT cells but not the LDLR−/−and LRP1−/−cells using microscopic analysis for cell morphology. The scale bar represents 50 μm. b The sensitivities of the U-87 MG WT, LDLR−/−, and LRP1−/−cells to Tcnα were quantified using the cytopathic cell rounding assay. The percentage of round-shaped cells was measured and plotted on the chart. Error bars (n = 6) indicate mean ± SD. The CR50 for Tcnα in the U-87 MG WT, LDLR−/−, and LRP1−/−cells were calculated and listed. c The indicated U-87 MG cells were transfected with mock, Ldlr, or LRP1CII-LdlrC, followed by incubation with Tcnα for 20 h. The percentages of round-shaped cells are plotted on the chart. Error bars (n = 6) indicate mean ± SD. d The indicated U-87 MG cells were incubated with Tcnα (30 pM, 20 h) and the images were captured. Representative images are shown. Red fluorescence (mCherry) marked transfected cells. The scale bar represents 50 μm.

Article Snippet: The following antibodies, reagents, and recombinant proteins were purchased from the indicated vendors: Alexa Fluor 488 goat anti-rabbit IgG (ab150077, 1:1000, Abcam), rabbit polyclonal IgG against β-Actin (ab227387, 1:5000, Abcam), rabbit monoclonal IgG against LDLR (ab52818 for western blot, 1:500; ab30532 for immunofluorescence, 1:200; Abcam), rabbit monoclonal IgG against LRP1 (ab92544, 1:20000 for western blot and 1:200 for immunofluorescence, Abcam), HRP-conjugated goat anti-human IgG-Fc antibody (SSA001, 1:3000, Sino Biological), Hoechst 33258 staining solution (E607301, BBI), NHS-Rhodamine fluorescent labeling kit (#46406, Thermo Fisher Scientific), recombinant human LRP1 Cluster II Fc chimera (R&D Systems, 2368-L2), Precast PAGE Gel (abs9309, Absin), Polyethylenimine Linear (PEI) MW25000 (40816ES03, YEASEN), and Heparin-Sepharose (Abcam, ab193268).

Techniques: Incubation, Transfection