rab11fip2  (Santa Cruz Biotechnology)

Journal: Cell Proliferation

Article Title: Alteration in Rab11‐mediated endocytic trafficking of LDL receptor contributes to angiotensin II ‐induced cholesterol accumulation and injury in podocytes

doi: 10.1111/cpr.13229

Figure Lengend Snippet: Expression alteration of LDLR and Rab11 in the glomeruli from Ang II‐infused mice. (A) Representative immunohistochemistry label of LDLR and Rab11 in kidney sections of control and Ang II infused mice. (original magnification ×40). (B) Western blot analysis of LDLR and Rab11 from glomerular lysates of control and Ang II treated mice. GAPDH was used as an equal loading marker, and the graph indicates a statistical result of relative protein levels. * p < 0.05, n = 5. (C and D) Fluorescence staining of LDLR or Rab11 (green) with Synaptopodin (a marker of podocytes, red), DAPI (nucleus, blue) to evaluate the expression levels (scale bar: 20 μm). (E) Quantitative RT‐PCR analysis to detect relative gene expressions in glomeruli from different groups. * p < 0.05, ** p < 0.01, n = 5. DAPI, 4′,6‐diamidino‐2‐phenylindole; LDLR, low‐density lipoprotein receptor

Article Snippet: For Ang II stimulation, podocytes were treated with Ang II (10 −7 M) for 24 h. For lysosome inhibition, podocytes were incubated with Leupeptin (20 mΜ, Topscience), an inhibitor of lysosomal protein degradation, for 30 min. For knockdown treatment, small interfering RNAs (siRNAs) targeting Rab11 (Qiagen) were transfected with HiPerFect (Qiagen) according to the manufacturer's instructions.

Techniques: Expressing, Immunohistochemistry, Control, Western Blot, Marker, Fluorescence, Staining, Quantitative RT-PCR

Journal: Cell Proliferation

Article Title: Alteration in Rab11‐mediated endocytic trafficking of LDL receptor contributes to angiotensin II ‐induced cholesterol accumulation and injury in podocytes

doi: 10.1111/cpr.13229

Figure Lengend Snippet: Enhanced interaction between LDLR and Rab11 in response to Ang II. (A) Western blot analysis of LDLR and Rab11 expression in HPCs exposed to Ang II, GAPDH was used as an equal loading marker, and the graph indicates a statistical result of relative protein levels. * p < 0.05, ** p < 0.01, n = 5. (B) Quantitative RT‐PCR analysis to detect relative gene expressions in HPCs from different groups. * p < 0.05, *** p < 0.001, n = 5. (C) Co‐IP with anti‐Rab11 antibody or an IgG negative control. The resulting precipitates, as well as a portion of the whole cell lysate, were subjected to immunoblotting with anti‐LDLR, Anti‐Rab11, and anti‐GAPDH antibodies. HPCs, human podocytes; IP, immunoprecipitation; LDLR, low‐density lipoprotein receptor

Article Snippet: For Ang II stimulation, podocytes were treated with Ang II (10 −7 M) for 24 h. For lysosome inhibition, podocytes were incubated with Leupeptin (20 mΜ, Topscience), an inhibitor of lysosomal protein degradation, for 30 min. For knockdown treatment, small interfering RNAs (siRNAs) targeting Rab11 (Qiagen) were transfected with HiPerFect (Qiagen) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Marker, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation

Journal: Cell Proliferation

Article Title: Alteration in Rab11‐mediated endocytic trafficking of LDL receptor contributes to angiotensin II ‐induced cholesterol accumulation and injury in podocytes

doi: 10.1111/cpr.13229

Figure Lengend Snippet: Ang II activated LDL recycling through Rab11. (A and B) Fluorescence staining of LDLR (green) with Rab11 (red) or Lamp1 (red) in HPCs (scale bar: 5 μm), and the graph indicates a statistical result of the average immunofluorescence intensity. Pearsonʼs coefficient and Mander's overlap coefficient are considered to represent the degree of panel colocalization. *** p < 0.001, ns = no significance, n = 30. HPCs, human podocytes; LDLR, low‐density lipoprotein receptor

Article Snippet: For Ang II stimulation, podocytes were treated with Ang II (10 −7 M) for 24 h. For lysosome inhibition, podocytes were incubated with Leupeptin (20 mΜ, Topscience), an inhibitor of lysosomal protein degradation, for 30 min. For knockdown treatment, small interfering RNAs (siRNAs) targeting Rab11 (Qiagen) were transfected with HiPerFect (Qiagen) according to the manufacturer's instructions.

Techniques: Fluorescence, Staining, Immunofluorescence

Journal: Cell Proliferation

Article Title: Alteration in Rab11‐mediated endocytic trafficking of LDL receptor contributes to angiotensin II ‐induced cholesterol accumulation and injury in podocytes

doi: 10.1111/cpr.13229

Figure Lengend Snippet: Rab11 knockdown reduced LDLR recycling and increased LDLR‐lysosomal degradation. HPCs were transfected with siRNAs targeting Rab11 (siRab11) or a scrambled control (Scramble) and then exposure to Ang II for 24 h. (A) Western blot analysis of total LDLR and Rab11 expression in HPCs, GAPDH was used as an equal loading marker, and the graph indicates a statistical result of relative protein levels. ** p < 0.01, *** p < 0.001, n = 5. (B and C) Fluorescence staining of LDLR (green) with Rab11 (red) or Lamp1 (red) in HPCs (scale bar: 5 μm), quantification was performed as described in Figure . ** p < 0.01, *** p < 0.001, n = 30. HPCs, human podocytes; LDLR, low‐density lipoprotein receptor

Article Snippet: For Ang II stimulation, podocytes were treated with Ang II (10 −7 M) for 24 h. For lysosome inhibition, podocytes were incubated with Leupeptin (20 mΜ, Topscience), an inhibitor of lysosomal protein degradation, for 30 min. For knockdown treatment, small interfering RNAs (siRNAs) targeting Rab11 (Qiagen) were transfected with HiPerFect (Qiagen) according to the manufacturer's instructions.

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Marker, Fluorescence, Staining

Journal: Cell Proliferation

Article Title: Alteration in Rab11‐mediated endocytic trafficking of LDL receptor contributes to angiotensin II ‐induced cholesterol accumulation and injury in podocytes

doi: 10.1111/cpr.13229

Figure Lengend Snippet: Rab11 inhibition reduced cholesterol and lipid droplets (LDs) content and protected podocytes from injury. (A) Representative confocal microscopy images and quantification of adipocyte differentiation‐related protein (Adrp, a marker of LDs) fluorescence staining and Filipin staining in each group. *** p < 0.001, n = 30. (B) Quantitative analysis of cholesterol content in each group. * p < 0.05, n = 6. (C) Flow cytometry analysis of podocytes apoptosis in different groups. * p < 0.05, ** p < 0.01, n = 6

Article Snippet: For Ang II stimulation, podocytes were treated with Ang II (10 −7 M) for 24 h. For lysosome inhibition, podocytes were incubated with Leupeptin (20 mΜ, Topscience), an inhibitor of lysosomal protein degradation, for 30 min. For knockdown treatment, small interfering RNAs (siRNAs) targeting Rab11 (Qiagen) were transfected with HiPerFect (Qiagen) according to the manufacturer's instructions.

Techniques: Inhibition, Confocal Microscopy, Marker, Fluorescence, Staining, Flow Cytometry