125 i egf  (Revvity)

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: EGF Internalization in AP2-KO MEFs or upon AP2-KD in Different Cell Contexts (A) 125 I-EGF (left) and 125 I-Tf (right) internalization in AP2-WT and AP2-KO MEFs in the presence or absence of clathrin KD. Internalization constants ( K e ) are the mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test ( ∗∗∗ p < 0.001). (B) Analysis of the impact of AP2μ KD versus clathrin KD in different cell lines as indicated. The number of EGFRs per cell in the different cell lines was measured using 125 I-EGF saturation binding assay. Kinetics of 125 I-EGF (1.5 ng/mL) were measured and are reported as internalization constants ( K e ). The percentage of AP2-independent EGFR internalization was calculated from the residual K e in AP2-KD cells relative to the K e in control cells (after subtracting the residual internalization in clathrin KD cells; Sigismund et al., 2013 ). (C) TEM analysis of EGFR internalization. AP2-WT and AP2-KO MEFs expressing EGFR under a doxycycline-inducible promoter (AP2-EGFR) were induced with doxycycline. Cells were then subjected to in vivo immunolabeling with anti-EGFR 13A9 antibody and 10 nm protein A-gold, stimulated 5 min with EGF (30 ng/mL), and fixed in the presence of ruthenium red, to distinguish PM-connected CCPs (ruthenium red positive) and internalized CCVs (ruthenium red negative) structures. Bar, 100 nm. (D) Morphometrical analysis of (C). Left: CCP number was normalized for the difference in PM length between AP2-KO MEFs versus control (∼1.5-fold increase; see Figure S3 A and ) and expressed as relative to control cells. Right: number of gold particles per CCS (CCPs + CCVs). N represents the numbers of random images (left) and CCSs (right) analyzed. Data are expressed as mean ± SEM. p values were calculated using two-tailed Student’s t test ( ∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Saturation Assay, Expressing, In Vivo, Immunolabeling, Two Tailed Test

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: Mechanism of AP2-Independent EGFR-CME: Role of eps15/L1 and epsin1 (A) Transient KD of the μ subunit of the indicated AP complexes was performed in AP2-KO MEFs, and K e of 125 I-EGF internalization was calculated. Data are mean ± SD (two replicates). (B) K e of 125 I-EGF internalization in stable eps15/eps15L1-KD HeLa cells transiently depleted of epsin1, alone or in combination with AP2μ, in comparison with AP2-KD and clathrin-KD HeLa cells. Results are mean ± SD of two to eight independent experiments. p values were calculated using each pair Student’s t test ( ∗∗ p < 0.01 and ∗∗∗ p < 0.001). (C) AP2-WT and AP2-KO MEFs were transiently depleted as indicated. K e values of 125 I-EGF internalization are shown as mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test ( ∗∗ p < 0.01 and ∗∗∗ p < 0.001). (D) TEM analysis of CCSs in AP2-WT-EGFR MEFs, control or triple KD for eps15, eps15L1, and epsin1, induced with doxycycline (to allow EGFR expression) and stimulated with EGF (30 ng/mL). Morphometrical analysis was performed on EGFR gold-positive structures. Left: CCP number was normalized for the difference in PM length between eps15/eps15L1/epsin1 KD MEFs versus control (∼0.8 decrease; see Figure S3 A and ) and expressed relative to control cells. Right: mean number of gold particle per CCS (CCPs + CCVs). N represents the numbers of random images (left panel) and numbers of CCSs (center and right panels) analyzed. Data are expressed as mean ± SEM. p values were calculated using each pair Student’s t test ( ∗ p < 0.05). (E) Automated analysis of clathrin-coated structure formation at the plasma membrane from 196 traces containing CLTA-TagRFP and AP2σ-EGFP (clathrin+/AP2+; left panel), 200 traces devoid of AP2σ-EGFP (clathrin+/AP2−; middle panel) from 10 MEF AP2-WT/triple-KD, or 154 traces from 10 MEF AP2-KO/triple-KD cells (right panel).

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Expressing

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: EGF-Dependent Signaling and Migration in AP2-Depleted Cells (A) HeLa cells were subjected to AP2 KD or eps15/L1/epsin1 KD followed by 125 I-EGF degradation assay at low EGF concentration (1.5 ng/mL; see ). At the indicated time points, (1) degraded EGF (top) represents the TCA soluble fraction of 125 I-EGF recovered in the medium and intracellularly, and (2) recycled EGF (bottom) represents the TCA insoluble fraction of 125 I-EGF recovered in the medium. Results are mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test ( ∗ p < 0.05 and ∗∗ p < 0.01). (B) Top: AP2-WT and AP2-KO MEFs were stimulated with low dose EGF (1.5 ng/mL) for the indicated times. Lysates were subjected to IB with the indicated antibodies. Bottom: quantitation of phosphoAKT signal normalized to total AKT (pAKT/AKT) and represented as percentage of signal in WT cells at 5 min of EGF stimulation. Results are mean ± SD of three independent experiments. p values were calculated using two-tailed Student’s t test ( ∗ p < 0.05 versus WT at each time point). (C) Left: HeLa cells were subjected to AP2 KD and/or eps15/L1/epsin1 KD in different combinations, followed by stimulation with low dose EGF (1.5 ng/mL) for the indicated times. IB was as shown. Right: quantitation of phosphoAKT signal normalized to total AKT (pAKT/AKT) and represented as percentage of signal in control cells at 5 min of EGF stimulation. Results are mean ± SD of three independent experiments. p values were calculated using two-tailed Student’s t test. ∗ p < 0.05 versus control at each time point. (D) Transwell migration assay of AP2 KD and eps15/L1/epsin1 KD HeLa cells under serum-starved (SS) conditions or in the presence of low-dose EGF (1.5 ng/mL) or 10% serum as indicated. Right: quantitation of migrating cells per field. Results are the mean ± SD of two to four independent experiments. p values were calculated using two-tailed Student’s t test ( ∗ p < 0.05 versus control in each condition).

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Migration, Degradation Assay, Concentration Assay, Quantitation Assay, Two Tailed Test, Transwell Migration Assay

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet:

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Transduction, Recombinant, Electron Microscopy, TaqMan Assay, Plasmid Preparation, Software