Journal: Heliyon

Article Title: Structure-based virtual screening of natural compounds in preventing skin senescence: The role of epigallocatechin gallate in protein kinase C alpha-specific inhibition against UV-induced photoaging

doi: 10.1016/j.heliyon.2024.e39933

Figure Lengend Snippet: In silico and in vitro , EGCG specifically inhibits PKCα by direct binding. ( a ) Classification and structural characteristics of protein kinase C family. ( b–d ) Binding affinity Gscore of the PKCα, PKCδ and PKCζ on EGCG. Binding affinities were measured by Schrodinger's software as described in the Materials and Methods. ( e ) EGCG directly binds to intracellular PKCα. The binding of EGCG with PKCα was performed SDS-PAGE and immunoblotting with a specific PKCα antibody: Lane 1, 2 (input control), cell lysates from HaCaT; Lane 3 (control), lysates from HaCaT; and Lane 4, cell lysates from HaCaT cells precipitated with EGCG-Sepharose 4B beads. ( f ) Effects of EGCG on PKCα kinase activity. Kinase activity was measured as described in the Materials and Methods. ### p < 0.001, ∗∗∗ p < 0.001 .

Article Snippet: Scrambled small interfering RNA (siRNA) (sc-37007) and PKCα siRNA (sc-36243) were procured from Santa Cruz Biotechnology.

Techniques: In Silico, In Vitro, Binding Assay, Software, SDS Page, Western Blot, Control, Activity Assay

Journal: Heliyon

Article Title: Structure-based virtual screening of natural compounds in preventing skin senescence: The role of epigallocatechin gallate in protein kinase C alpha-specific inhibition against UV-induced photoaging

doi: 10.1016/j.heliyon.2024.e39933

Figure Lengend Snippet: Knockdown of PKCα inhibits UVB-induced MMP-1 expression. ( a ) In HaCaT cells, transfecting PKCα with siRNA resulted in a significant knockdown of PKCα protein expression and attenuated MMP-1 expression was determined by western blotting analysis and quantified with Image J software. ( b ) The mRNA levels of MMP-1 were detected using RT-qPCR. ### p < 0.001, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Scrambled small interfering RNA (siRNA) (sc-37007) and PKCα siRNA (sc-36243) were procured from Santa Cruz Biotechnology.

Techniques: Knockdown, Expressing, Western Blot, Software, Quantitative RT-PCR

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Expression of insulin‐like growth factor‐binding protein 5 (IGFBP5) is elevated in ischaemic tissue or hypoxic human umbilical vein endothelial cells (HUVECs). (A) Representative immunofluorescence images and (B) quantification of the expression of IGFBP5 in ischaemic or non‐ischaemic tissues from patients with critical limb ischaemia ( n = 6). (C) Representative immunofluorescence images and (D) quantification of the expression of IGFBP5 in gastrocnemius of the ischaemic hindlimb of mice ( n = 5). (E) mRNA expression of IGFBP1‐7 tested by qRT‐PCR in HUVECs under hypoxia stimulation for 24 h ( n = 6). (F) Representative Western blotting images and quantification of the expression of IGFBP5 in HUVECs under hypoxia stimulation for 24 h. (G) Representative immunofluorescence images and (H) quantification of the expression of IGFBP5 in HUVECs under hypoxia stimulation for 24 h ( n = 6).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Expressing, Binding Assay, Immunofluorescence, Quantitative RT-PCR, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Endothelial cell (EC)‐specific deletion of insulin‐like growth factor‐binding protein 5 (IGFBP5) attenuates the damage of the ischaemic hindlimb in mice. (A) Representative blood flow images of laser Doppler‐based tissue perfusion system in CDH5‐Cre‐IGFBP5 flox/flox (IGFBP5 EKO ) and IGFBP5 flox/flox (IGFBP5 f/f ) mice after ischaemia induction (0 W) and 4 weeks after ischaemia. (B) Quantification of blood flow in the hindlimb of IGFBP5 EKO and IGFBP5 f/f mice before (−1 D) and after ischaemia induction at 0, 1, 2, 3 and 4 weeks ( n = 8 in each group). ### p < .001 compared to IGFBP5 f/f ‐sham; * p < .05, ** p < .01 and *** p < .001 compared to IGFBP5 f/f ‐HLI. (C) Percentage of limb salvage, foot necrosis and limb loss in IGFBP5 EKO and IGFBP5 f/f mice treated with sham or hindlimb ischaemia (HLI) ( n = 8 in each group). (D) Representative immunofluorescence staining images and (E) quantification of CD31 (red) in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (F) Representative haematoxylin‒eosin and picrosirius red staining images and (G) quantification of fibrosis area in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (H) Representative immunofluorescence staining images and (I) quantification of dihydroethidium (DHE) staining for reactive oxygen species (ROS) in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (J) Representative immunofluorescence staining images and (K) quantification of TUNEL staining in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (L) Representative microscopy images of aortic sprouting assay and (M) quantification of tube length of the aorta from IGFBP5 EKO and IGFBP5 f/f mice at 4 and 7 days after being placed in plates with Matrigel. (N) Representative images and (O) quantification of cutaneous wound healing in IGFBP5 EKO and IGFBP5 f/f mice at 0, 4 and 7 days after wound induction ( * p < .05, *** p < .001 vs. IGFBP5 f/f ).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Immunofluorescence, Staining, TUNEL Assay, Microscopy

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Insulin‐like growth factor‐binding protein 5 (IGFBP5) knockdown promotes tube formation, cell proliferation and migration in human umbilical vein endothelial cells (HUVECs). (A) Representative heatmap, (B) volcano plot and (C) Gene Ontology (GO) biological process enrichment pathway in IGFBP5 shRNA (sh‐IGFBP5)‐ or control shRNA (sh‐NC)‐transfected HUVECs. (D) Representative images and (E) quantification of tube formation in sh‐NC‐ and sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (F) Representative immunofluorescence images and (G) quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU, green)‐stained HUVECs transfected with sh‐NC or sh‐IGFBP5 ( n = 5 in each group). (H) Representative images and (I) quantification of flow cytometry for cell cycle in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (J) Representative images and (K) quantification of wound healing assay in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (L) Representative images and (M) quantification of transwell assay in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (N) Representative Western blotting images and (O) quantification of IGFBP5, p‐IGF1R and IGF1R expression in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs. (P) Representative Western blotting images and (Q) quantification of p‐Erk1/2, Erk1/2, p‐Akt and Akt expression in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 6 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Knockdown, Migration, shRNA, Control, Transfection, Immunofluorescence, Staining, Flow Cytometry, Wound Healing Assay, Transwell Assay, Western Blot, Expressing

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Recombinant human insulin‐like growth factor‐binding protein 5 (IGFBP5) suppresses tube formation, cell proliferation and migration. (A) Representative images and (B) quantification of tube formation in recombinant human IGFBP5 (rhIGFBP5)‐ or vehicle‐treated human umbilical vein endothelial cells (HUVECs). (C) Representative immunofluorescence images and (G) quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU, green)‐stained HUVECs treated with rhIGFBP5 or vehicle ( n = 5 in each group). (E) Representative images and (F) quantification of flow cytometry for cell cycle in rhIGFBP5‐ or vehicle‐treated HUVECs ( n = 5 in each group). (G) Representative images and (H) quantification of wound healing assay in rhIGFBP5‐ or vehicle‐treated HUVECs. (I) Representative images and (J) quantification of transwell assay in rhIGFBP5‐ or vehicle‐treated HUVECs ( n = 5 in each group). Representative Western blotting images and quantification of (K) p‐IGF1R and IGF1R, (L) p‐Erk1/2 and Erk1/2 and (M) p‐Akt, and Akt in rhIGFBP5‐ or vehicle‐treated HUVECs ( n = 6 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Recombinant, Binding Assay, Migration, Immunofluorescence, Staining, Flow Cytometry, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Recombinant human insulin‐like growth factor‐binding protein 5 (IGFBP5) restrains IGF1‐ or IGF2‐induced tube formation, cell proliferation and migration. (A) Representative tube formation images and quantification of human umbilical vein endothelial cells (HUVECs) treated with rhIGF1 in the presence or absence of recombinant human IGFBP5 (rhIGFBP5) ( n = 5 in each group). (B) Representative tube formation images and quantification of HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (C) Representative immunofluorescence images and quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU, green)‐stained HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (D) Representative immunofluorescence images and quantification of EdU (green)‐stained HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (E) Representative images and quantification of flow cytometry for cell cycle of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (F) Representative images and quantification of flow cytometry for cell cycle of HUVECs treated with rhIGF2 in the presence or absence of rhGFBP5 ( n = 5 in each group). (G) Representative images and quantification of wound healing assay of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (H) Representative images and quantification of wound healing assay of HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (I) Representative images and quantification of transwell assay of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (J) Representative images and quantification of transwell assay of HUVECs treated with rhIGF2 in the presence or absence of hIGFBP5 ( n = 5 in each group). (K) Representative images of Western blotting assay‐detected time‐course of p‐IGF1R, IGF1R, p‐Erk1/2, Erk1/2, p‐Ak and Akt expression of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5. (L) Representative images of Western blotting assay‐detected time‐course of p‐IGF1R, IGF1R, p‐Erk1/2, Erk1/2, p‐Ak and Akt expression of HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5.

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Recombinant, Binding Assay, Migration, Immunofluorescence, Staining, Flow Cytometry, Wound Healing Assay, Transwell Assay, Western Blot, Expressing

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Insulin‐like growth factor‐binding protein 5 (IGFBP5) deficiency promotes glycolysis through IGF1R. (A) OCR and (B) ECAR profiles in sh‐NC‐ and sh‐IGFBP5‐infected human umbilical vein endothelial cells (HUVECs) ( n = 8 in each group). (C) Quantification of ATP production in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs. (D) OCR and (E) ECAR profiles showing the glycolytic function in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 8 in each group). (F) Quantification of glycolytic function parameters in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 8 in each group). (G) Western blotting images and (H) quantification of the expression of lactate dehydrogenase (LDH), PKM1 and PKM2 in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs. (I) OCR, (J) ECAR, (K) proton efflux rate (PER) and (L) glycoPER profiles showing glycolytic function in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor picropodophyllin (PPP) ( n = 6 in each group). (M) Quantification of glycolytic function parameters in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of PPP ( n = 8 in each group). (N) Western blot images and (O) quantification of the expression of LDH, PKM1 and PKM2 in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor PPP ( n = 6 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Infection, Western Blot, Expressing

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Deletion of insulin‐like growth factor‐binding protein 5 (IGFBP5) increases HIF1α expression by suppressing ubiquitination via ubiquitin ligase Von Hippel‐Lindau (VHL). (A) Western blotting images and quantification of HIF1α in sh‐NC‐ and sh‐IGFBP5‐infected human umbilical vein endothelial cells (HUVECs) ( n = 6 in each group). (B) Western blotting images and quantification of HIF1α in NC‐overexpression (OE‐NC)‐ and IGFBP5‐overexpression (OE‐IGFBP5)‐transfected HUVECs ( n = 6 in each group). (C) The stability of HIF1α expression in sh‐NC‐ and sh‐IGFBP5‐transfected HUVECs in the presence of 20 μg/mL cycloheximide (CHX) determined by Western blot ( n = 5 in each group). (D) The stability of HIF1α expression in OE‐NC‐ and OE‐IGFBP5‐transfected HUVECs in the presence of MG132 (MG) determined by Western blot. (E) Western blotting images of total ubiquitin, K48 ubiquitin and K63 ubiquitin of HIF1α in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 3). (F) Western blotting images and quantification of VHL in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 5 in each group). (G) Co‐immunoprecipitation of the interaction between VHL and HIF1α detected by Western blot in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 3). (H) Western blot images and quantification of HIF1α expression in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor picropodophyllin (PPP) ( n = 5 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Expressing, Ubiquitin Proteomics, Western Blot, Infection, Over Expression, Transfection, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Endothelial cell (EC)‐specific deletion of IGF1R abolished IGFBP5 EKO ‐attenuated limb ischaemia. (A) Representative images detected by laser Doppler‐based tissue perfusion system in IGFBP5 EKO mice, which were infected with AAV2/9‐sh‐NC or AAV‐sh‐IGF1R after ischaemia induction (0 W) and 4 weeks after ischaemia (4 W). (B) Quantification of blood flow in the hindlimb of IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV‐sh‐IGF1R before (−1 D) and after ischaemia induction at 0 days, and 1, 2, 3 and 4 weeks ( n = 8 in each group). (C) Percentage of limb salvage, foot necrosis and limb loss in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 8 in each group). (D) Representative images of immunofluorescence staining, and quantification of CD31 (red) and DAPI (blue) in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group). (E) Representative images of haematoxylin‒eosin and picrosirius red staining, and quantification of fibrosis area in gastrocnemius of the hindlimb in ischaemic or sham in IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group). (F) Representative immunofluorescence staining images and quantification of dihydroethidium (DHE) staining for reactive oxygen species (ROS) detection in gastrocnemius of the hindlimb in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group). (G) Representative immunofluorescence staining images and quantification of TUNEL staining in gastrocnemius of the hindlimb in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Infection, Immunofluorescence, Staining, TUNEL Assay

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Schematic illustration describing the underlying mechanism of insulin‐like growth factor‐binding protein 5 (IGFBP5)‐regulated endothelial cell angiogenesis post‐hindlimb ischaemia (HLI). After HLI, IGFBP5 production was promoted, which inhibited the action of IGF1/2 and the phosphorylation of IGF1R and ATP production, thereby promoting the ubiquitin‐ligase Von Hippel‐Lindau (VHL) and the ubiquitination and degradation of HIF1α. When deficiency of IGFBP5 in endothelial cells (ECs), the IGF1R phosphorylation was promoted, the ATP production was promoted, and the ubiquitination of HIF1α was suppressed, which promoted angiogenesis post‐HLI.

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Phospho-proteomics, Ubiquitin Proteomics