Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Intracellular distribution of LDLRs revealed by immunofluorescence staining (red) or ectopic expression of GFP‐LDLR (green) in unstimulated NIH‐3T3 and HepG2 cells. Cell nuclei were counterstained with DAPI (outlined by the dotted lines). Double fluorescence labeling assays illustrating that LDLRs exhibited partial co‐localization with Rab11 and Rab4a in the perinuclear region (indicated by arrowheads) in unstimulated cells. Results of fluorescence intensity profile analysis (original images shown in Appendix Fig ) showing that AAS (50%) treatment induced a redistribution of LDLRs toward the cell periphery in NIH‐3T3 cells. Note the increased red/green signals distal to the nuclei (DAPI). Con, complete culture medium. Double fluorescence labeling assays illustrating that AAS increased LDLR co‐localization with EEA1, Rab4a, and Rab5 (mainly in the peripheral region) (arrowheads), whereas LDLRs displayed little co‐localization with Rab7 or Vps35 either without or with AAS treatment. Fluorescence intensity profile analysis (sampling locations as indicated by the dotted lines in panel D) showing that AAS increased LDLR co‐localization with Rab4a in the cell periphery. Results of surface biotin‐disulfide pulse‐chase labeling experiments showing that AAS accelerated the mobilization of LDLRs for both of the entry into and the exit from the intracellular space (mean data from 3 independent experiments). PC, positive control (sample omitting the disulfide cleavage step); NC, negative control (sample omitting the biotinylation step). Fluorescence microscopy data showing that overexpressing dominant negative Rab4a‐S22N suppressed AAS‐stimulated DiI‐LDL endocytosis, whereas overexpressing constitutively active Rab4a‐Q67L mimicked the effect of AAS in resting cells. Overexpressing WT Rab4a enhanced AAS‐induced response but had no effect in unstimulated cells. Double fluorescence labeling experiments showing that, in unstimulated cells, Rab4a‐Q67L exhibited co‐localization with LDLRs in both of the perinuclear region and the cell periphery (arrowheads), whereas Rab4a‐S22N only co‐localized with LDLRs in the perinuclear region. Double immunofluorescence labeling showing that there was not a perinuclear pool for transferrin receptor (TfR) in unstimulated cells. Although TfRs also showed co‐localization with Rab4a throughout the cytosol, this pattern of distribution was not affected by AAS treatment. Fluorescence microscopy data showing that AAS did not increase the rate of endocytosis of FITC‐labeled transferrin. Representative double fluorescence labeling results illustrating that there was not a significant perinuclear co‐localization between Rab11 and TfR in unstimulated cells. FRAP assay results in the cells expressing GFP‐LDLR showing that fluorescence recovery in the putative perinuclear pool was slower than that in the peripheral region. The quantitative data shown below were average values from 8–10 individual cells. Inhibitory effects of gene silencing for WASH1 and COMMD1 on the basal and AAS‐stimulated LDL uptake. Left panels, representative western blots showing gene silencing efficacy of the siRNA constructs (from 3 independent experiments with similar results). Right panel, quantitative DiI fluorescence microscopy data showing that WASH1 and COMMD1 gene silencing reduced the basal level of LDL uptake and also abolished the response induced by AAS. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA. NS, no significance. All immunofluorescence labeling experiments were repeated at least three times. Experiments in (B) to (J) were performed in NIH‐3T3 cells; (K) to (M) in HepG2 cells. Nu, nucleus. Source data are available online for this figure.

Article Snippet: Antibodies for the following targets were used: LDLR (10785‐1‐AP, RRID:AB_2281164) (for western blot and immunofluorescence), Na‐K ATPase (14418‐1‐AP, RRID:AB_2227873), LC3 (14600‐1‐AP, RRID:AB_2137737), Rab11 (20229‐1‐AP, RRID:AB_10666202), GFP (50430‐2‐AP, RRID:AB_11042881), GDI 1 (10249‐1‐AP, RRID:AB_2111520), Rabenosyn‐5 (22218‐1‐AP, RRID:AB_11182179), p62/SQSTM1 (18420‐1‐AP, RRID:AB_10694431), vinculin (66305‐1‐Ig, RRID:AB_2810300), megalin/LDL receptor‐related protein 2 (LRP2) (19700‐1‐AP, RRID:AB_10640428) (for immunofluorescence), β‐actin (66009‐1‐Ig), GAPDH (60004‐1‐Ig), and COMMD1 (11938‐1‐AP, RRID:AB_2083542) were from Proteintech (Wuhan, Hubei Province, China).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Labeling, Sampling, Pulse Chase, Positive Control, Negative Control, Microscopy, Dominant Negative Mutation, FRAP Assay, Western Blot, Construct

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Immunoprecipitation (IP) and western blot (WB) experiments in cells expressing GFP‐Rab4a fusion protein showing that AAS did not change the interaction between Rab4a and GDI. H/chain, Ig heavy chain. Normal IgG was used as negative control for IP. Double fluorescence labeling experiment showing that AAS had no effect on Rab4a‐GDI co‐localization (experiment repeated 3 times). Sequence alignment of the putative CaMKII substrate site (Thr137) in Rab4a across different species. In vitro phosphorylation experiments using FLAG‐tagged wild type (WT) and T137A mutant of Rab4a(120‐154) peptide showing that CaMKII could phosphorylate the wild‐type peptide (experiment repeated 2 times). p‐(S/T), anti‐phospho serine/threonine antibody. Representative results of IP/WB experiments in NIH‐3T3 cells expressing wild‐type GFP‐Rab4a or GFP‐Rab4a‐T137A, showing that AAS increased the phosphorylation of wild‐type Rab4a but not Rab4a‐T137A. Representative results of IP/WB experiments showing that AAS increased the phosphorylation level of endogenous Rab4a, and this response was abolished by KN‐93 pretreatment. Representative fluorescence microscopy images showing that in the cells with a high level of Rab4a‐T137A expression (arrows), AAS failed to stimulate DiI‐LDL endocytosis, while in cells with poor Rab4a‐T137A expression (asterisk), AAS‐stimulated DiI‐LDL endocytosis was retained. Surface protein biotinylation assay showing that Rab4a‐T137A overexpression diminished the effect of AAS on LDLR accumulation on PM (example from 3 independent experiments with similar results). Western blots and quantitative data showing that treatment with the serine/threonine protein phosphatase inhibitor okadaic acid (OA, 0.5 μM for 60 min) increased the phosphorylation level of CaMKII (Thr286) in nonstressed cells. Fluorescence microscopy data showing that okadaic acid treatment in nonstressed cells mimicked the effect of AAS on DiI‐LDL endocytosis. Double fluorescence labeling and fluorescence intensity profile analysis showing that okadaic acid treatment in nonstressed cells increased LDLR‐Rab4a co‐localization in the cell periphery (arrowheads) (experiment repeated 3 times). Dotted lines indicated the sampling location for fluorescence intensity profile analysis. Representative IP/WB results showing that in cells overexpressing GFP‐Rab4a fusion proteins, AAS increased Rabenosyn‐5 binding with wild‐type Rab4a, but not that with Rab4a‐T137A. The stimulating effect of AAS on Rabenosyn‐5/Rab4a binding was blunted in the presence of KN‐93. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA or unpaired t ‐test as appropriate. Source data are available online for this figure.

Article Snippet: Antibodies for the following targets were used: LDLR (10785‐1‐AP, RRID:AB_2281164) (for western blot and immunofluorescence), Na‐K ATPase (14418‐1‐AP, RRID:AB_2227873), LC3 (14600‐1‐AP, RRID:AB_2137737), Rab11 (20229‐1‐AP, RRID:AB_10666202), GFP (50430‐2‐AP, RRID:AB_11042881), GDI 1 (10249‐1‐AP, RRID:AB_2111520), Rabenosyn‐5 (22218‐1‐AP, RRID:AB_11182179), p62/SQSTM1 (18420‐1‐AP, RRID:AB_10694431), vinculin (66305‐1‐Ig, RRID:AB_2810300), megalin/LDL receptor‐related protein 2 (LRP2) (19700‐1‐AP, RRID:AB_10640428) (for immunofluorescence), β‐actin (66009‐1‐Ig), GAPDH (60004‐1‐Ig), and COMMD1 (11938‐1‐AP, RRID:AB_2083542) were from Proteintech (Wuhan, Hubei Province, China).

Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Fluorescence, Labeling, Sequencing, In Vitro, Phospho-proteomics, Mutagenesis, Microscopy, Cell Surface Biotinylation Assay, Over Expression, Sampling, Binding Assay

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Intracellular distribution of LDLRs revealed by immunofluorescence staining (red) or ectopic expression of GFP‐LDLR (green) in unstimulated NIH‐3T3 and HepG2 cells. Cell nuclei were counterstained with DAPI (outlined by the dotted lines). Double fluorescence labeling assays illustrating that LDLRs exhibited partial co‐localization with Rab11 and Rab4a in the perinuclear region (indicated by arrowheads) in unstimulated cells. Results of fluorescence intensity profile analysis (original images shown in Appendix Fig ) showing that AAS (50%) treatment induced a redistribution of LDLRs toward the cell periphery in NIH‐3T3 cells. Note the increased red/green signals distal to the nuclei (DAPI). Con, complete culture medium. Double fluorescence labeling assays illustrating that AAS increased LDLR co‐localization with EEA1, Rab4a, and Rab5 (mainly in the peripheral region) (arrowheads), whereas LDLRs displayed little co‐localization with Rab7 or Vps35 either without or with AAS treatment. Fluorescence intensity profile analysis (sampling locations as indicated by the dotted lines in panel D) showing that AAS increased LDLR co‐localization with Rab4a in the cell periphery. Results of surface biotin‐disulfide pulse‐chase labeling experiments showing that AAS accelerated the mobilization of LDLRs for both of the entry into and the exit from the intracellular space (mean data from 3 independent experiments). PC, positive control (sample omitting the disulfide cleavage step); NC, negative control (sample omitting the biotinylation step). Fluorescence microscopy data showing that overexpressing dominant negative Rab4a‐S22N suppressed AAS‐stimulated DiI‐LDL endocytosis, whereas overexpressing constitutively active Rab4a‐Q67L mimicked the effect of AAS in resting cells. Overexpressing WT Rab4a enhanced AAS‐induced response but had no effect in unstimulated cells. Double fluorescence labeling experiments showing that, in unstimulated cells, Rab4a‐Q67L exhibited co‐localization with LDLRs in both of the perinuclear region and the cell periphery (arrowheads), whereas Rab4a‐S22N only co‐localized with LDLRs in the perinuclear region. Double immunofluorescence labeling showing that there was not a perinuclear pool for transferrin receptor (TfR) in unstimulated cells. Although TfRs also showed co‐localization with Rab4a throughout the cytosol, this pattern of distribution was not affected by AAS treatment. Fluorescence microscopy data showing that AAS did not increase the rate of endocytosis of FITC‐labeled transferrin. Representative double fluorescence labeling results illustrating that there was not a significant perinuclear co‐localization between Rab11 and TfR in unstimulated cells. FRAP assay results in the cells expressing GFP‐LDLR showing that fluorescence recovery in the putative perinuclear pool was slower than that in the peripheral region. The quantitative data shown below were average values from 8–10 individual cells. Inhibitory effects of gene silencing for WASH1 and COMMD1 on the basal and AAS‐stimulated LDL uptake. Left panels, representative western blots showing gene silencing efficacy of the siRNA constructs (from 3 independent experiments with similar results). Right panel, quantitative DiI fluorescence microscopy data showing that WASH1 and COMMD1 gene silencing reduced the basal level of LDL uptake and also abolished the response induced by AAS. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA. NS, no significance. All immunofluorescence labeling experiments were repeated at least three times. Experiments in (B) to (J) were performed in NIH‐3T3 cells; (K) to (M) in HepG2 cells. Nu, nucleus. Source data are available online for this figure.

Article Snippet: Antibodies for the following targets were used: LDLR (10785‐1‐AP, RRID:AB_2281164) (for western blot and immunofluorescence), Na‐K ATPase (14418‐1‐AP, RRID:AB_2227873), LC3 (14600‐1‐AP, RRID:AB_2137737), Rab11 (20229‐1‐AP, RRID:AB_10666202), GFP (50430‐2‐AP, RRID:AB_11042881), GDI 1 (10249‐1‐AP, RRID:AB_2111520), Rabenosyn‐5 (22218‐1‐AP, RRID:AB_11182179), p62/SQSTM1 (18420‐1‐AP, RRID:AB_10694431), vinculin (66305‐1‐Ig, RRID:AB_2810300), megalin/LDL receptor‐related protein 2 (LRP2) (19700‐1‐AP, RRID:AB_10640428) (for immunofluorescence), β‐actin (66009‐1‐Ig), GAPDH (60004‐1‐Ig), and COMMD1 (11938‐1‐AP, RRID:AB_2083542) were from Proteintech (Wuhan, Hubei Province, China).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Labeling, Sampling, Pulse Chase, Positive Control, Negative Control, Microscopy, Dominant Negative Mutation, FRAP Assay, Western Blot, Construct

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Preliminary western blot analysis of samples from the cell surface protein biotinylation and pull‐down assay (example from 2 independent experiments with similar results) showing that only a minor fraction of LDLRs (red arrows) were located on the surface of plasma membrane (eluate). Na‐K ATPase was used as a membrane marker. Samples omitting the biotinylation step (Biotin −) were included as negative control. Experiments were performed in NIH‐3T3 cells. Western blots and quantitative densitometry data showing that AAS increased the amount of LDLRs on plasma membrane. The eluate samples obtained as indicated in panel A were further concentrated before western blotting. Flow cytometry with nonpermeabilized NIH‐3T3 cells showing that AAS increased the abundance of LDLRs on the cell surface. The quantitative data were shown on the right. Anti‐LDLR antibody pulse labeling followed by flow cytometry showing that the detected PM surface LDLRs in both resting and AAS‐treated cells participated in the endocytic turnover. In the left panel, the cells were pulse‐labeled with unconjugated LDLR antibody at 4°C, then returned to 37°C for 60 min, and finally labeled with FITC‐conjugated secondary antibody (2° Ab) at 4°C. In the right panel, the cells were pulse‐labeled with PE‐conjugated primary antibody using the same protocol, and flow cytometry was carried out without secondary antibody labeling. Isotype Ig was used as negative control. Data shown were example from 3 independent experiments with similar results. Real‐time PCR results showing that AAS had no significant effects on LDLR mRNA levels in NIH‐3T3 and HepG2 cells. Western blots and quantitative densitometry data showing that AAS had no significant effects on the total LDLR protein levels. Fluorescence microscopy data showing that pretreatment with actinomycin D (ActD, 10 μg/ml) or cycloheximide (Cyclo, 30 μg/ml) in NIH‐3T3 cells did not change AAS (50%)‐stimulated DiI‐LDL endocytosis. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA. NS, no significance. Source data are available online for this figure.

Article Snippet: LDLR (AF2148, RRID:AB_2135126) (unconjugated goat IgG, for double immunofluorescence labeling of endogenous LDLR), LDLR (FAB2148P, RRID:AB_10573833) (PE‐conjugated, for flow cytometry), megalin/LRP2 (FAB9578G) (for flow cytometry), and human apolipoprotein B (ApoB) (AF3556, RRID:AB_573025) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Pull Down Assay, Clinical Proteomics, Membrane, Marker, Negative Control, Flow Cytometry, Labeling, Antibody Labeling, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Intracellular distribution of LDLRs revealed by immunofluorescence staining (red) or ectopic expression of GFP‐LDLR (green) in unstimulated NIH‐3T3 and HepG2 cells. Cell nuclei were counterstained with DAPI (outlined by the dotted lines). Double fluorescence labeling assays illustrating that LDLRs exhibited partial co‐localization with Rab11 and Rab4a in the perinuclear region (indicated by arrowheads) in unstimulated cells. Results of fluorescence intensity profile analysis (original images shown in Appendix Fig ) showing that AAS (50%) treatment induced a redistribution of LDLRs toward the cell periphery in NIH‐3T3 cells. Note the increased red/green signals distal to the nuclei (DAPI). Con, complete culture medium. Double fluorescence labeling assays illustrating that AAS increased LDLR co‐localization with EEA1, Rab4a, and Rab5 (mainly in the peripheral region) (arrowheads), whereas LDLRs displayed little co‐localization with Rab7 or Vps35 either without or with AAS treatment. Fluorescence intensity profile analysis (sampling locations as indicated by the dotted lines in panel D) showing that AAS increased LDLR co‐localization with Rab4a in the cell periphery. Results of surface biotin‐disulfide pulse‐chase labeling experiments showing that AAS accelerated the mobilization of LDLRs for both of the entry into and the exit from the intracellular space (mean data from 3 independent experiments). PC, positive control (sample omitting the disulfide cleavage step); NC, negative control (sample omitting the biotinylation step). Fluorescence microscopy data showing that overexpressing dominant negative Rab4a‐S22N suppressed AAS‐stimulated DiI‐LDL endocytosis, whereas overexpressing constitutively active Rab4a‐Q67L mimicked the effect of AAS in resting cells. Overexpressing WT Rab4a enhanced AAS‐induced response but had no effect in unstimulated cells. Double fluorescence labeling experiments showing that, in unstimulated cells, Rab4a‐Q67L exhibited co‐localization with LDLRs in both of the perinuclear region and the cell periphery (arrowheads), whereas Rab4a‐S22N only co‐localized with LDLRs in the perinuclear region. Double immunofluorescence labeling showing that there was not a perinuclear pool for transferrin receptor (TfR) in unstimulated cells. Although TfRs also showed co‐localization with Rab4a throughout the cytosol, this pattern of distribution was not affected by AAS treatment. Fluorescence microscopy data showing that AAS did not increase the rate of endocytosis of FITC‐labeled transferrin. Representative double fluorescence labeling results illustrating that there was not a significant perinuclear co‐localization between Rab11 and TfR in unstimulated cells. FRAP assay results in the cells expressing GFP‐LDLR showing that fluorescence recovery in the putative perinuclear pool was slower than that in the peripheral region. The quantitative data shown below were average values from 8–10 individual cells. Inhibitory effects of gene silencing for WASH1 and COMMD1 on the basal and AAS‐stimulated LDL uptake. Left panels, representative western blots showing gene silencing efficacy of the siRNA constructs (from 3 independent experiments with similar results). Right panel, quantitative DiI fluorescence microscopy data showing that WASH1 and COMMD1 gene silencing reduced the basal level of LDL uptake and also abolished the response induced by AAS. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA. NS, no significance. All immunofluorescence labeling experiments were repeated at least three times. Experiments in (B) to (J) were performed in NIH‐3T3 cells; (K) to (M) in HepG2 cells. Nu, nucleus. Source data are available online for this figure.

Article Snippet: LDLR (AF2148, RRID:AB_2135126) (unconjugated goat IgG, for double immunofluorescence labeling of endogenous LDLR), LDLR (FAB2148P, RRID:AB_10573833) (PE‐conjugated, for flow cytometry), megalin/LRP2 (FAB9578G) (for flow cytometry), and human apolipoprotein B (ApoB) (AF3556, RRID:AB_573025) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Labeling, Sampling, Pulse Chase, Positive Control, Negative Control, Microscopy, Dominant Negative Mutation, FRAP Assay, Western Blot, Construct

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Immunoprecipitation (IP) and western blot (WB) experiments in cells expressing GFP‐Rab4a fusion protein showing that AAS did not change the interaction between Rab4a and GDI. H/chain, Ig heavy chain. Normal IgG was used as negative control for IP. Double fluorescence labeling experiment showing that AAS had no effect on Rab4a‐GDI co‐localization (experiment repeated 3 times). Sequence alignment of the putative CaMKII substrate site (Thr137) in Rab4a across different species. In vitro phosphorylation experiments using FLAG‐tagged wild type (WT) and T137A mutant of Rab4a(120‐154) peptide showing that CaMKII could phosphorylate the wild‐type peptide (experiment repeated 2 times). p‐(S/T), anti‐phospho serine/threonine antibody. Representative results of IP/WB experiments in NIH‐3T3 cells expressing wild‐type GFP‐Rab4a or GFP‐Rab4a‐T137A, showing that AAS increased the phosphorylation of wild‐type Rab4a but not Rab4a‐T137A. Representative results of IP/WB experiments showing that AAS increased the phosphorylation level of endogenous Rab4a, and this response was abolished by KN‐93 pretreatment. Representative fluorescence microscopy images showing that in the cells with a high level of Rab4a‐T137A expression (arrows), AAS failed to stimulate DiI‐LDL endocytosis, while in cells with poor Rab4a‐T137A expression (asterisk), AAS‐stimulated DiI‐LDL endocytosis was retained. Surface protein biotinylation assay showing that Rab4a‐T137A overexpression diminished the effect of AAS on LDLR accumulation on PM (example from 3 independent experiments with similar results). Western blots and quantitative data showing that treatment with the serine/threonine protein phosphatase inhibitor okadaic acid (OA, 0.5 μM for 60 min) increased the phosphorylation level of CaMKII (Thr286) in nonstressed cells. Fluorescence microscopy data showing that okadaic acid treatment in nonstressed cells mimicked the effect of AAS on DiI‐LDL endocytosis. Double fluorescence labeling and fluorescence intensity profile analysis showing that okadaic acid treatment in nonstressed cells increased LDLR‐Rab4a co‐localization in the cell periphery (arrowheads) (experiment repeated 3 times). Dotted lines indicated the sampling location for fluorescence intensity profile analysis. Representative IP/WB results showing that in cells overexpressing GFP‐Rab4a fusion proteins, AAS increased Rabenosyn‐5 binding with wild‐type Rab4a, but not that with Rab4a‐T137A. The stimulating effect of AAS on Rabenosyn‐5/Rab4a binding was blunted in the presence of KN‐93. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA or unpaired t ‐test as appropriate. Source data are available online for this figure.

Article Snippet: LDLR (AF2148, RRID:AB_2135126) (unconjugated goat IgG, for double immunofluorescence labeling of endogenous LDLR), LDLR (FAB2148P, RRID:AB_10573833) (PE‐conjugated, for flow cytometry), megalin/LRP2 (FAB9578G) (for flow cytometry), and human apolipoprotein B (ApoB) (AF3556, RRID:AB_573025) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Fluorescence, Labeling, Sequencing, In Vitro, Phospho-proteomics, Mutagenesis, Microscopy, Cell Surface Biotinylation Assay, Over Expression, Sampling, Binding Assay

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Western blots and quantitative densitometry data showing that AAS‐induced autophagic response was delayed by addition of exogenous LDL (100 μg/ml) in HepG2 cells. Real‐time PCR results showing that AAS‐induced autophagic responses were delayed by exogenous LDL in HepG2 cells ( n = 4–5 independent experiments). Immunofluorescence data showing that exogenous LDL blunted AAS‐induced LC3 puncta formation in HepG2 cells. Immunofluorescence data showing that exogenous LDL blunted AAS‐induced LC3 puncta formation in WT but not LDLR −/− primary hepatocytes. Western blot results showing that Pitstop2 (20 μM) and KN‐93 (5 μM) abolished the compensatory effect of exogenous LDL on AAS‐induced autophagic response in HepG2 cells. Western blot results showing that in NIH‐3T3 cells, the compensatory effect of exogenous LDL on autophagy induction in response to AAS was attenuated by overexpression of Rab4a‐T137A. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA (except for the data in (E) and (F), which were examined with nonparametric Kruskal–Wallis test because of non‐Gaussian distribution). Source data are available online for this figure.

Article Snippet: LDLR (AF2148, RRID:AB_2135126) (unconjugated goat IgG, for double immunofluorescence labeling of endogenous LDLR), LDLR (FAB2148P, RRID:AB_10573833) (PE‐conjugated, for flow cytometry), megalin/LRP2 (FAB9578G) (for flow cytometry), and human apolipoprotein B (ApoB) (AF3556, RRID:AB_573025) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Over Expression

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Roles of different Ras downstream effectors in the induction of Fra-1. (A) Scheme of the activity of the Ras (V12) effector mutants. The C40, G37, and S35 effector loop mutations enable the Ras (V12) oncoprotein to activate selectively the PI3-kinase (C40)-, the RalGDS (G37)-, or the Raf (S35)-dependent cascade. (B) Immunoblotting and RT-PCR analysis of the activity of Ras (V12) effector mutants on the expression of fra-1 in thyroid cells. The fra-1/β-globin reporter construct (10 μg) was cotransfected with the empty vector (pCDNA3) or the indicated Ras expression vector (5 μg) in FRTL-5 cells. As a control of activity in the transformed cell line, the reporter construct was transfected in FRTL-5Kras cells. The total DNA was kept to 20 μg, and 3 μg of the pCMV-CAT reporter was cotransfected as an internal control for transfection efficiency. After 36 h cell extracts or total RNA was prepared. For immunoblotting (upper panel), 50 μg of cell extracts was processed as described in Materials and Methods and was probed with anti-Fra-1 antibody (Santa Cruz Biotechnology, Inc.). As a control for equal loading, the blotted proteins were stained with Red-Ponceau (not shown). For the RT-PCR (lower panel), total RNA was reverse transcribed and the 76-bp β-globin transcript was coamplified with the 346-bp CAT mRNA in the presence of [α32-P]dCTP and was analyzed by 5% PAGE. (C) Diagram of PhosphorImager quantitation (ImageQuant software) of the RT-PCR data. The relative activity of the Ras effector double mutants is expressed as a percentage of the activity of the Ras (V12) construct which resulted in the maximal stimulation of the fra-1/β-globin reporter. In the right-hand diagram the results are shown as fold induction of the reporter gene relative to its activity in FRTL-5 cells. These experiments were repeated three times with similar results. (D) In vitro MBP phosphorylation assay of ERK activation by Ras (V12) effector mutants. The Ras expression constructs or the empty vector were cotransfected in FRTL-5 cells along with the vector encoding the epitope-tagged ERK2 (pcDNA3-ERK2-HA). After 24 h cells were collected and equal amounts of cell lysates were immunoprecipitated (IP) with α-HA antibody or nonimmune serum and were subjected to in vitro phosphorylation reaction as described in Materials and Methods. Reaction products were analyzed by SDS-10% PAGE.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Plasmid Preparation, Transformation Assay, Transfection, Staining, Quantitation Assay, Software, In Vitro, Phosphorylation Assay, Activation Assay, Immunoprecipitation

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Effect of chemical inhibition of the MEK/ERK pathway on Fra-1 expression. FRTL-5KRas cells were treated with 10 μM U0126 (Promega) for 4, 8, or 12 h. Total RNA or whole-cell extracts were prepared from untreated FRTL-5 (lane 1) and FRTL-5KRas (lane 2) cells or from FRTL-5KRas cells treated with U0126 (lanes 3 to 5). (A) For Northern blotting, 30 μg of total RNA was analyzed by hybridization to the radiolabeled rat fra-1 cDNA probe as indicated in Materials and Methods. The ethidium bromide staining of rRNAs (28S and 18S) was utilized as a control for RNA loading. (B) For immunoblotting analysis, 50-μg samples of cell extracts were processed as described in Materials and Methods. The same membrane was first incubated with the α-Fra-1 antibody and subsequently was stripped and reprobed with α-P-ERK1/2 (New England Biolabs) as a control for the inhibitory activity of the drug. Finally, the blot was incubated with α-ERK1 antibody (New England Biolabs) as a control for equal protein loading. For the in vitro dephosphorylation reaction (B, right-hand panel) the cell extracts from untreated FRTL-5KRas cells were incubated for 2 h at 37°C with or without 20 U of CIP prior to SDS-10% PAGE and immunoblotting. Northern blotting and immunoblotting data were confirmed by multiple experiments, and similar results were obtained by using the PD 98059 MEK inhibitor.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Inhibition, Expressing, Northern Blot, Hybridization, Staining, Western Blot, Incubation, Activity Assay, In Vitro, De-Phosphorylation Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Effect of chemical inhibition of the MEK/ERK pathway on Fra-1 stability and DNA binding activity. (A) Pulse-chase analysis of the Fra-1 half-life in FRTL-5KRas cells treated with the MEK inhibitor. Cells pretreated for 30 min with 10 μM U0126 were subjected to pulse-chase labeling, as described in Materials and Methods, alone or in the presence of U0126. Control cells were treated with the vehicle (dimethyl sulfoxide). Cells were collected at the indicated time points. Whole-cell extracts were immunoprecipitated with the α-Fra-1 antibody and were analyzed by SDS-PAGE. −ab, without antibody. (B) Diagram of densitometric quantitation of the results (QuantityOne software) showing the kinetics of decay of distinct Fra-1 electrophoretic isoforms in untreated cells (upper, middle, and lower bands) compared to that of U0126-treated cells (middle and lower bands) expressed as the relative optical density of the autoradiographic image. Similar results were obtained in four different pulse-chase experiments. (C) Immunoblotting analysis of Fra-1 following DNA affinity chromatography of nuclear extracts subjected to in vitro dephosphorylation. The upper left-most panel shows 25 μg of nuclear extract from FRTL-5KRas cells incubated with 5 μg of fra-1 TRE DNA-agarose beads in the absence of competitor oligonucleotide (no competitor) or after preincubation with a 25-fold molar excess of fra-1 TRE or mut fra-1 TRE competitor oligonucleotides. The eluted TRE-bound material was subjected to SDS-10% PAGE along with the same amount of untreated proteins (input). The two upper right panels show 25 μg of nuclear extract subjected to in vitro dephosphorylation (+CIP) and compared to the untreated control (−CIP). The lower panel is on off-rate analysis of an equivalent untreated sample (25 μg) subjected to DNA affinity chromatography, as described in Materials and Methods. A 25-fold molar excess of competitor fra-1 TRE oligonucleotide was added, and the DNA-agarose-bound complex was allowed to dissociate at room temperature during the indicated time course prior to elution and SDS-PAGE. The diagram was obtained by quantitation of the chemiluminescence signal by use of the Gel-Doc image acquisition apparatus and QuantityOne software (Bio-Rad). The data, representing the average of two independent experiments, were normalized by attributing the 100% value to the first time point for each of the two electrophoretic isoforms (upper and lower). IP, immunoprecipitation.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Inhibition, Binding Assay, Activity Assay, Pulse Chase, Labeling, Immunoprecipitation, SDS Page, Quantitation Assay, Software, Western Blot, Affinity Chromatography, In Vitro, De-Phosphorylation Assay, Incubation

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Characterization of the Ras-responsive element of the fra-1 gene. (A) Schematic representation of the fra-1/β-globin reporter constructs. The −710 to +2741 region of the rat fra-1 gene was fused to a portion of the rabbit β-globin gene (exons 2 and 3, black arrow) to generate a stable chimeric transcript. The fra-1-derived sequence includes the (−710) promoter region, the first exon, the first intron, and part of the second exon (white boxes). The first intron contains two AP-1-like sites (empty circles) and an AP-1 consensus element (fra-1 TRE, black circle) deleted in the mutated version (fra-1/β-globin-Δ). For stable clones, FRTL-5 and FRTL-5KRas cell lines were transfected with the DNA vector encoding the selectable marker (pCMV-Neo), alone (MOCK clones) or in combination with the linearized fra-1/β-globin wild type (wt) or the fra-1/β-globin-Δ construct. After selection in G418 (800 μg/ml; Calbiochem), pools of stably transfected cell clones were derived from a similar number (∼80 to 100) of G418-resistant colonies. The comparable copy number of stably integrated constructs in each pool of G418-resistant transfectants was verified by Southern blot hybridization with a rabbit β-globin probe detecting an internal 3.8-kb EcoRI fragment (data not shown). (B) RT-PCR analysis of the basal and serum-inducible expression of the chimeric transcript in the FRTL-5- and FRTL-5KRas-derived pools of transfected cell clones. Cells were maintained in normal growth conditions before stimulation with 20% fetal calf serum for 3 h. Total RNA was prepared from MOCK cell clones (lanes 1 and 6) and from cell clones expressing the wild type (lanes 2 to 3 and 7 to 8) or the deletion-containing (lanes 4 to 5 and 9 to 10) chimeric minigene. For RT-PCR, 2 μg of DNase-treated RNA was reverse transcribed as described in Materials and Methods and the 76-bp β-globin cDNA fragment was amplified together with the 370-bp HPRT cDNA as an internal control. PCR products were resolved by 4% agarose gel electrophoresis. (C and D) Serum induction of fra-1 and mRNA and protein binding to fra-1 TRE. FRTL-5 and FRTL-5KRas cells were maintained in complete medium or were switched into a medium containing 0.5% FBS for 48 h, and then 20% FBS was added 3 h prior to harvest. Total RNA or nuclear proteins were extracted from cycling (lanes 1 and 4), serum-arrested (lanes 2 and 5), and serum-stimulated (lanes 3 and 6) cells. (C) For Northern blot analysis, 30 μg of total RNA/sample was hybridized to the radiolabeled rat fra-1 cDNA probe, as indicated in Materials and Methods. Equal loading was verified by ethidium bromide staining of rRNAs (bottom panel). (D) EMSA of the serum-induced binding to the fra-1 TRE. Nuclear proteins were incubated with the 5′-end-labeled fra-1 TRE oligonucleotide before 5% polyacrylamide gel retardation. The arrows indicate the oligoncleotide/AP-1 complex. All results were confirmed in at least three independent experiments.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Construct, Derivative Assay, Sequencing, Clone Assay, Transfection, Plasmid Preparation, Marker, Selection, Stable Transfection, Southern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Agarose Gel Electrophoresis, Protein Binding, Northern Blot, Staining, Binding Assay, Incubation, Labeling, Electrophoretic Mobility Shift Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: In vitro analysis of the fra-1 TRE binding complex in the cell clones expressing the MEK and Rac constitutive derivatives. (A) EMSA of AP-1 binding to the fra-1 TRE oligonucleotide in the FRTL-5-derived cell clones expressing the constitutively active form of MEK and/or Rac (FRTL-5MEK, FRTL-5Rac, and FRTL-5MEK/Rac). Nuclear proteins (3 μg) were incubated with the labeled fra-1 TRE oligonucleotide before PAGE. (B) Immunoblotting analysis of Fra-1 and Jun proteins in the normal (FRTL-5), Ha-ras- or Ki-ras-transformed (FRTL-5Hras and FRTL-5KRas), and stably transfected cell lines (FRTL-5MEK, FRTL-5Rac, and FRTL-5MEK/Rac). Nuclear proteins were separated by SDS-PAGE (20 μg/lane) and were transferred to a polyvinylidene difluoride membrane. Western blots were sequentially incubated with anti-Fra-1, anti-c-Jun, anti-JunB, and anti-JunD followed by anti-α-tubulin antibodies as a control for equal loading. The arrows indicate the major isoforms of Fra-1 (38 kDa), c-Jun (39 kDa), and JunB (38 kDa) and the two isoforms of JunD (41 and 37 kDa). (C) Antibody supershift analysis of the complex bound to the fra-1 TRE oligonucleotide. After protein binding to the labeled oligonucleotide, nuclear extracts were incubated for 3 h with the indicated antibodies before gel retardation. Different autoradiographic exposures were chosen for different panels (FRTL-5, 24 h; FRTL-5MEK and FRTL-5 MEK/Rac, 16 h; FRTL-5Kras, 8 h) to allow the optimal visualization of supershifted complexes. The arrows indicate the AP-1/oligonucleotide complex, while the asterisks refer to the supershifted ternary complexes. In vitro binding and supershift assays were repeated at least twice with comparable results.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: In Vitro, Binding Assay, Clone Assay, Expressing, Derivative Assay, Incubation, Labeling, Western Blot, Transformation Assay, Stable Transfection, Transfection, SDS Page, Protein Binding, Electrophoretic Mobility Shift Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: In vivo occupancy of the fra-1 TRE in normal and ras-transformed cells. (A) Radioactive PCR on naked DNA from normal and transformed cells as a control for the amplification products. Chromosomal DNA extracted from FRTL-5 and FRTL-5KRas was analyzed by radioactive PCR. The fra-1-Int primers amplified a 594-bp DNA region containing the intronic fra-1 TRE, while the primers for the HPRT gene gave rise to a 558-bp PCR product. (B) ChIp of the +178- to +772-nucleotide region encompassing the fra-1 TRE. Following in vivo formaldehyde cross-linking, chromatin extracted from FRTL-5 and FRTL-5KRas cells was immunoprecipitated with anti-Fra-1 or anti-acetylated histone H3 antibodies (α-AcH3). Following reversal of cross-linking, the purified DNA fragments were amplified by radioactive PCR with the fra-1-Int and HPRT primers as a control for equal input. The products were resolved by native gel electrophoresis and were detected by autoradiography. (C) The radioactive signal of the amplification products was quantified by PhosphorImager (with ImageQuant software), normalized for the HPRT internal control and expressed as relative to the no-antibody (−Ab)/FRTL-5 control sample. The reproducibility of these results was confirmed by repeating the ChIp experiments two times independently.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: In Vivo, Transformation Assay, Amplification, Immunoprecipitation, Purification, Nucleic Acid Electrophoresis, Autoradiography, Software

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Analysis of the Fra-1-dependent transactivation in response to the ras oncogene and transactivation activity of Gal4/Fra-1 fusion proteins. The reporter plasmid FrLuc (5 μg) was coexpressed with the vectors (10 μg) expressing the Gal4/Fra-1, GAL4/Fra-1-ΔZip, GAL4/c-Fos, or GAL4/c-Fos-insZip chimeric protein, along with the pCDNA3-Ras (V12) expression vector (5 μg) or the pCDNA3 empty vector. As an internal control, a vector encoding Renilla Luciferase (0.5 μg) was cotransfected. Thirty-six hours after transfection cells were harvested and assayed for both firefly and Renilla luciferase activities (Dual-Luciferase Reporter Assay system) to allow for normalizing of the FrLuc reporter activity for variations of transfection efficiency. The diagram shows the relative luciferase activity in the absence (hatched boxes) or presence (black boxes) of pCDNA3-Ras (V12). The results represent the average of three independent experiments, with the error bars indicating the standard errors.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Luciferase, Transfection, Reporter Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Model of multistep positive autoregulation of Fra-1 in ras-transformed cells. The thickness of the arrows originating from the constitutively GTP-bound oncogene (Ras*) indicates the relative contribution of different Ras-dependent pathways in the activation of the fra-1 promoter. Following ERK-dependent phosphorylation, Fra-1 is stabilized in transformed cells and interacts with Jun family partners. The Jun/Fra-1 heterodimer can activate fra-1 gene transcription by binding the autoregulatory site in its first intron (fra-1 TRE) and recruiting a transcriptional coactivator (CBP/p300). White boxes, fra-1 exons; thick black line, fra-1 5′-flanking and first intron; grey line, fra-1 TRE; trash bin, degradation apparatus.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Transformation Assay, Activation Assay, Binding Assay

Journal: EMBO Reports

Article Title: Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

doi: 10.15252/embr.202153373

Figure Lengend Snippet: Intracellular distribution of LDLRs revealed by immunofluorescence staining (red) or ectopic expression of GFP‐LDLR (green) in unstimulated NIH‐3T3 and HepG2 cells. Cell nuclei were counterstained with DAPI (outlined by the dotted lines). Double fluorescence labeling assays illustrating that LDLRs exhibited partial co‐localization with Rab11 and Rab4a in the perinuclear region (indicated by arrowheads) in unstimulated cells. Results of fluorescence intensity profile analysis (original images shown in Appendix Fig ) showing that AAS (50%) treatment induced a redistribution of LDLRs toward the cell periphery in NIH‐3T3 cells. Note the increased red/green signals distal to the nuclei (DAPI). Con, complete culture medium. Double fluorescence labeling assays illustrating that AAS increased LDLR co‐localization with EEA1, Rab4a, and Rab5 (mainly in the peripheral region) (arrowheads), whereas LDLRs displayed little co‐localization with Rab7 or Vps35 either without or with AAS treatment. Fluorescence intensity profile analysis (sampling locations as indicated by the dotted lines in panel D) showing that AAS increased LDLR co‐localization with Rab4a in the cell periphery. Results of surface biotin‐disulfide pulse‐chase labeling experiments showing that AAS accelerated the mobilization of LDLRs for both of the entry into and the exit from the intracellular space (mean data from 3 independent experiments). PC, positive control (sample omitting the disulfide cleavage step); NC, negative control (sample omitting the biotinylation step). Fluorescence microscopy data showing that overexpressing dominant negative Rab4a‐S22N suppressed AAS‐stimulated DiI‐LDL endocytosis, whereas overexpressing constitutively active Rab4a‐Q67L mimicked the effect of AAS in resting cells. Overexpressing WT Rab4a enhanced AAS‐induced response but had no effect in unstimulated cells. Double fluorescence labeling experiments showing that, in unstimulated cells, Rab4a‐Q67L exhibited co‐localization with LDLRs in both of the perinuclear region and the cell periphery (arrowheads), whereas Rab4a‐S22N only co‐localized with LDLRs in the perinuclear region. Double immunofluorescence labeling showing that there was not a perinuclear pool for transferrin receptor (TfR) in unstimulated cells. Although TfRs also showed co‐localization with Rab4a throughout the cytosol, this pattern of distribution was not affected by AAS treatment. Fluorescence microscopy data showing that AAS did not increase the rate of endocytosis of FITC‐labeled transferrin. Representative double fluorescence labeling results illustrating that there was not a significant perinuclear co‐localization between Rab11 and TfR in unstimulated cells. FRAP assay results in the cells expressing GFP‐LDLR showing that fluorescence recovery in the putative perinuclear pool was slower than that in the peripheral region. The quantitative data shown below were average values from 8–10 individual cells. Inhibitory effects of gene silencing for WASH1 and COMMD1 on the basal and AAS‐stimulated LDL uptake. Left panels, representative western blots showing gene silencing efficacy of the siRNA constructs (from 3 independent experiments with similar results). Right panel, quantitative DiI fluorescence microscopy data showing that WASH1 and COMMD1 gene silencing reduced the basal level of LDL uptake and also abolished the response induced by AAS. Data information: Data were mean ± SD. * P < 0.05, one‐way ANOVA. NS, no significance. All immunofluorescence labeling experiments were repeated at least three times. Experiments in (B) to (J) were performed in NIH‐3T3 cells; (K) to (M) in HepG2 cells. Nu, nucleus. Source data are available online for this figure.

Article Snippet: Antibodies for the following targets were used: LDLR (10785‐1‐AP, RRID:AB_2281164) (for western blot and immunofluorescence), Na‐K ATPase (14418‐1‐AP, RRID:AB_2227873), LC3 (14600‐1‐AP, RRID:AB_2137737), Rab11 (20229‐1‐AP, RRID:AB_10666202), GFP (50430‐2‐AP, RRID:AB_11042881), GDI 1 (10249‐1‐AP, RRID:AB_2111520), Rabenosyn‐5 (22218‐1‐AP, RRID:AB_11182179), p62/SQSTM1 (18420‐1‐AP, RRID:AB_10694431), vinculin (66305‐1‐Ig, RRID:AB_2810300), megalin/LDL receptor‐related protein 2 (LRP2) (19700‐1‐AP, RRID:AB_10640428) (for immunofluorescence), β‐actin (66009‐1‐Ig), GAPDH (60004‐1‐Ig), and COMMD1 (11938‐1‐AP, RRID:AB_2083542) were from Proteintech (Wuhan, Hubei Province, China).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Labeling, Sampling, Pulse Chase, Positive Control, Negative Control, Microscopy, Dominant Negative Mutation, FRAP Assay, Western Blot, Construct