Journal: bioRxiv

Article Title: Probability-based detection of phosphoproteomic uncertainty reveals rare signaling events driven by oncogenic kinase gene fusion

doi: 10.1101/621961

Figure Lengend Snippet: Hierarchical clustering of Markov-Chain Monte Carlo modelled data using a Euclidian distance metric distinctly clustered the fusion-kinase expressing cells from control cells, and fusion-kinase expressing cells grown in standard cell culture from those grown in colony formation assays. The concentration of quantified proteins ( A ), and the occupancy of quantified phosphorylation sites ( B ) were expressed as log ratios to thyroid cells expressing Cas9.

Article Snippet: The following day, cells were transfected with pCMV-VSV-G, psPAX2, and pCW-Cas9 vectors using MirusBio TransIT-LT1 transfection reagent.

Techniques: Expressing, Cell Culture, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination

doi: 10.1084/jem.20151673

Figure Lengend Snippet: Overexpression of FBXL14 promoted GSC differentiation and inhibited GSC tumor growth. (A) IB analysis of FBXL14, c-Myc, SOX2, GFAP, and MAP2 in the GSCs transduced with Flag-FBXL14 expression through lentiviral infection during a time course of differentiation. Forced expression of FBXL14 in GSCs accelerated expression of differentiated cell markers (GFAP and MAP2) and augmented loss of GSC markers (c-Myc and SOX2) during serum-induced GSC differentiation. VEC, vector. (B–E) Immunofluorescent staining of Flag-FBXL14 and c-Myc (B) or GFAP (C) in GSCs (T387) expressing Flag-FBXL14 or vector control during a time course of GSC differentiation induced by serum (FBS). Cells were counterstained with DAPI (blue) to mark nuclei. Quantifications show that forced expression of FBXL14 significantly accelerated the reduction of c-Myc–positive cells (D) and increased percentage of GFAP-positive cells (E) during serum-induced GSC differentiation. n = 4. Data are from three independent experiments, and representative images are shown. (F–I) The effect of FBXL14 overexpression on GSC tumorsphere formation. GSCs (T387) were transduced with Flag-FBXL14 or vector control. (G) Ectopic expression of Flag-FBXL14 reduced tumorsphere formation of GSCs. (H and I) Quantifications show that forced expression of FBXL14 significantly decreased GSC tumorsphere size (H) and number (I). n = 3–4. Data are from three independent experiments, and representative images are shown. (J) Growth curves of GSCs expressing Flag-FBXL14 or vector control. GSCs (T387) were transduced with Flag-FBXL14 or vector control through lentiviral infection and then measured for cell growth over a time course. Ectopic expression of FBXL14 significantly inhibited GSC growth. n = 4. Data are from three independent experiments. Data are mean ± SD. ***, P < 0.001. Student’s t test was used to assess the significance. (A and F) Mass is shown in kilodaltons.

Article Snippet: The inducible overexpression vector pCW was obtained from Addgene.

Techniques: Over Expression, Transduction, Expressing, Infection, Plasmid Preparation, Staining

Journal: The Journal of Experimental Medicine

Article Title: Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination

doi: 10.1084/jem.20151673

Figure Lengend Snippet: Overexpression of FBXL14 inhibited GBM tumor growth and promoted animal survival. (A and B) In vivo bioluminescent imaging of GBM xenografts derived from luciferase-expressing GSCs transduced with FBXL14 or vector (VEC) control. GSCs (T387) were transduced with luciferase and Flag-FBXL14 or vector control and then transplanted into brains of immunocompromised mice (5 × 10 3 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (A) Representative images at the indicated days are shown. (B) Luminescence quantification indicated that ectopic expression of FBXL14 significantly attenuated GSC tumor growth in mouse brains. Data are mean ± SD. n = 5. ***, P < 0.001. Student’s t test was used to assess the significance. Five mice per group were used. (C) Representative images of mouse brain sections from mice intracranially implanted with the GSCs transduced with Flag-FBXL14 or vector control. GSCs (T387) were transduced with Flag-FBXL14 or vector control through lentiviral infection for 48 h and then transplanted into mouse brains. Cross sections (hematoxylin and eosin stained) of the mouse brains harvested on day 21 after injection are shown. (D) Kaplan-Meier survival curves of mice intracranially implanted with the GSCs expressing Flag-FBXL14 or vector control. Ectopic expression of FBXL14 significantly increased survival of mice bearing the GSC-derived xenografts. Log-rank analysis was used. Five mice per group were used. (E) IB analysis of FBXL14 and c-Myc in GSCs (T387) transduced with inducible FBXL14 (pCW-Flag-FBXL14) and then treated with doxycycline (Dox) for 3 d. FBXL14 overexpression decreased c-Myc protein levels. Mass is shown in kilodaltons. Ctrl, control. (F) Growth curves of GSCs transduced with inducible FBXL14 expression and treated with doxycycline or control. Cells were measured for cell growth over a time course (day 0 to day 8). Overexpression of FBXL14 significantly inhibited the growth of GSCs. Data are mean ± SD. n = 3. Student’s t test was used to assess the significance. Data are from three independent experiments. (G and H) In vivo bioluminescent imaging of GBM xenografts derived from the luciferase-labeled GSCs transduced with inducible FBXL14 overexpression and treated with doxycycline or control. GSCs (T387) were transduced with inducible FBXL14 expression (pCW-Flag-FBXL14) and then transplanted into brains of immunocompromised mice (10 4 cells per animal). Mice bearing the intracranial xenografts were closely monitored. 7 d after GSC transplantation, mice were treated with 2 mg/ml doxycycline in drinking water to induce expression of FBXL14 in xenografts. (G) Representative images at the indicated days are shown. (H) Bioluminescence quantification indicated that induced overexpression of FBXL14 attenuated GSC tumor growth in mouse brains at day 21. Data are mean ± SD. n = 5. Student’s t test was used to assess the significance. Five mice per group were used. (I) Kaplan-Meier survival curves of mice intracranially implanted with the GSCs transduced with pCW-Flag-FBXL14 for 7 d and then treated with doxycycline or control. Induced expression of FBXL14 significantly increased survival of mice bearing the GSC-derived xenografts. Log-rank analysis was used. Five mice per group were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The inducible overexpression vector pCW was obtained from Addgene.

Techniques: Over Expression, In Vivo, Imaging, Derivative Assay, Luciferase, Expressing, Transduction, Plasmid Preparation, Transplantation Assay, Infection, Staining, Injection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination

doi: 10.1084/jem.20151673

Figure Lengend Snippet: FBXL14 mediates ubiquitination and degradation of c-Myc, whereas USP13 stabilizes c-Myc protein through deubiquitination. (A) Ubiquitination (Ub) assay showing that overexpression of FBXL14 promoted degradation of c-Myc in GSCs. GSCs (T387) were transduced with Flag-FBXL14 or vector control through lentiviral infection for 48 h, treated with the proteasome inhibitor MG132 for 6 h, harvested for IP of c-Myc with an anti–c-Myc antibody, and then immunoblotted with anti-ubiquitin antibody. Total cell lysates (TCL) were also immunoblotted with antibodies against FBXL14, USP13, c-Myc, and tubulin. (B) Ubiquitination assay showing that FBXL14 knockdown decreased c-Myc ubiquitination in GSCs. GSCs (T387) were transduced with shFBXL14 or shNT through lentiviral infection for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collecting samples. Cell lysates were immunoprecipitated with an anti–c-Myc antibody and then immunoblotted with anti-ubiquitin antibody. (C) Ubiquitination assay showing that FBXL14 mediated ubiquitination of wild-type c-Myc (lane 4) but not the ubiquitin-insensitive c-Myc mutants (Mt): T58A-Myc (lane 5), S62A-Myc (lane 6), and T58A-S62A-Myc (lane 7). Flag-FBXL14 or vector control and wild-type c-Myc or the c-Myc mutant were overexpressed in 293FT cells and then subjected to ubiquitination assay and IB analysis. (D) Ubiquitination assay showing that USP13 knockdown increased c-Myc ubiquitination and decreased c-Myc protein levels in GSCs. GSCs (T387) were transduced with shUSP13 (shUSP13-50 or shUSP13-52) or shNT control for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collecting samples. Cell lysates were immunoprecipitated with an anti–c-Myc antibody and then immunoblotted with anti-ubiquitin antibody. (E) Ubiquitination assay showing that c-Myc ubiquitination was specifically attenuated by wild-type USP13 but not catalytic dead mutant USP13 (C345A). GSCs (T387 or T4121) were transduced with Flag-USP13, Flag-USP13-C345A, or vector control for 48 h, treated with the proteasome inhibitor MG132 for 6 h, and then subjected to analysis of c-Myc ubiquitination and IB analyses. (F) Ubiquitination analysis showing that USP13-mediated deubiquitination antagonizes FBXL14-mediated c-Myc ubiquitination to regulate c-Myc protein levels in GSCs. GSCs (T387) were transduced with HA-FBXL14, Flag-USP13, Flag-USP13 plus HA-FBXL14, or vector control for 2 d through lentiviral infection, treated with MG132 for 6 h, and harvested for ubiquitination assay of c-Myc and IB analyses. FBXL14 overexpression alone increased c-Myc ubiquitination (lane 3), whereas USP13 overexpression alone decreased c-Myc ubiquitination (lane 4). However, USP13 and FBXL14 double overexpression attenuated the increased c-Myc ubiquitination induced by FBXL14 overexpression (lane 5). (G) Ubiquitination assay showing that c-Myc protein level was regulated by USP13-mediated deubiquitination and FBXL14-induced ubiquitination in GSCs. GSCs (T387) were differentiated for 2 d and transduced with shUSP13, shFBXL14, shUSP13 plus shFBXL14, or shNT for 2 d through lentiviral infection, treated with MG132 for 6 h, and then collected for ubiquitination analysis and IB analyses. USP13 knockdown alone increased c-Myc ubiquitination (lane 3), whereas FBXL14 knockdown alone decreased c-Myc ubiquitination (lane 4). However, USP13 and FBXL14 double knockdown attenuated the increased c-Myc ubiquitination induced by USP13 knockdown (lane 5). Mass is shown in kilodaltons.

Article Snippet: The inducible overexpression vector pCW was obtained from Addgene.

Techniques: Over Expression, Transduction, Plasmid Preparation, Infection, Ubiquitin Assay, Immunoprecipitation, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination

doi: 10.1084/jem.20151673

Figure Lengend Snippet: T58A–c-Myc mutant rescued the phenotype caused by USP13 knockdown or FBXL14 overexpression. (A) IB analysis of Flag-T58A–c-Myc mutant, endogenous c-Myc (WT), USP13, FBXL14, GFAP, and SOX2 in the T387 GSCs transduced with Flag-T58A–c-Myc or vector (Vec) control in combination with shUSP13 or shNT control. USP13 knockdown decreased the endogenous wild-type c-Myc but not the ectopic Flag-T58A–c-Myc mutant. (B) IB analysis of Flag-FBXL14, HA-T58A–c-Myc mutant, endogenous c-Myc (WT), USP13, GFAP, and SOX2 in the GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control. Ectopic expression of FBXL14 decreased endogenous wild-type c-Myc but not the HA-T58A–c-Myc mutant. (A and B) Mass is shown in kilodaltons. (C–E) Tumorsphere formation of GSCs transduced with Flag-T58A–c-Myc or vector control in combination with shUSP13 or shNT control. (C) Representative images (phase contrast) of tumorspheres are shown. Ectopic expression of Flag-T58A–c-Myc mutant in GSCs rescued the impaired tumorsphere formation caused by USP13 disruption. (D and E) Quantification indicated that ectopic expression of the T58A–c-Myc mutant in GSCs rescued the decreased tumorsphere size (D) and number (E) caused by USP13 disruption. Data are mean ± SD. n = 4. ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (F–H) Tumorsphere formation of GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector (VEC) control. (F) Representative images (phase contrast) of tumorspheres are shown. Ectopic expression of the T58A–c-Myc mutant in GSCs rescued the reduced tumorsphere formation caused by FBXL14 overexpression. (G and H) Quantification indicated that ectopic expression of the T58A–c-Myc mutant in GSCs rescued the decreased tumorsphere size (G) and number (H) caused by FBXL14 overexpression. Data are mean ± SD. n = 4. ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (I and J) In vivo bioluminescent imaging of GBM xenografts derived from the luciferase-labeled GSCs transduced with Flag-T58A–c-Myc mutant or vector control in combination with shUSP13 or shNT control. The luciferase-labeled GSCs (T387) were transduced with Flag-T58A–c-Myc or vector control and shUSP13 or shNT through lentiviral infection and then transplanted into the brains of immunocompromised mice (5 × 10 3 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (I) Representative images at the indicated days are shown. (J) Quantification of luminescence indicates that ectopic expression of the T58A–c-Myc mutant restored the GSC tumorigenic potential impaired by USP13 disruption. Data are mean ± SD. n = 5. ***, P < 0.001; VEC + shUSP13 versus other groups. Student’s t test was used to assess the significance. Five mice per group were used. (K and L) In vivo bioluminescent imaging of GBM xenografts derived from luciferase-expressing GSCs transduced with HA-T58A–c-Myc mutant or vector control in combination with Flag-FBXL14 or vector control. GSCs (T387) were transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control through lentiviral infection and then transplanted into immunocompromised mouse brains (5 × 10 3 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (K) Representative images at the indicated days are shown. (L) Luminescence quantification shows that ectopic expression of the c-Myc–T58A mutant restored GSC tumorigenic potential attenuated by ectopic expression of FBXL14. Data are mean ± SD. n = 5. ***, P < 0.001; VEC + FBXL14 versus other groups. Student’s t test was used to assess the significance. Five mice per group were used. (M) Kaplan-Meier survival curves of mice implanted with GSCs transduced with Flag-T58A–c-Myc or vector control in combination with shUSP13 or shNT control. Mice implanted with the GSCs were maintained until the development of neurological signs or for 60 d. Ectopic expression of the T58A–c-Myc mutant in GSCs significantly attenuated the increased survival of mice caused by USP13 disruption in the GSC-derived tumors. ***, P < 0.001; VEC + shUSP13 versus other groups. Log-rank analysis was used. Five mice per group were used. (N) Kaplan-Meier survival curves of mice implanted with GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control. Mice implanted with the GSCs were maintained until the development of neurological signs or for 60 d. Ectopic expression of the T58A–c-Myc mutant in GSCs significantly attenuated the increased survival of mice caused by FBXL14 expression in the GSC-derived tumors. ***, P < 0.001; VEC + FBXL14 versus other groups. Log-rank analysis was used. Five mice per group were used.

Article Snippet: The inducible overexpression vector pCW was obtained from Addgene.

Techniques: Mutagenesis, Over Expression, Transduction, Plasmid Preparation, Expressing, In Vivo, Imaging, Derivative Assay, Luciferase, Labeling, Infection, Transplantation Assay

Journal: bioRxiv

Article Title: MicroRNA-focused CRISPR/Cas9 Screen Identifies miR-142 as a Key Regulator of Epstein-Barr Virus Reactivation

doi: 10.1101/2024.01.15.575629

Figure Lengend Snippet: A. Schematic of the miRNA-focused CRISPR/Cas9 loss-of- function screen carried out in EBV-positive MutuI-iCas9 BL cells. B. DrugZ-calculated normZ score plotted versus miRNA gene rank. Red indicates targets that were significantly enriched in gp350-positive population while black indicates targets significantly enriched in the gp350- negative population (p-value < 0.05). Highlighted is miR-142 which showed the most significant changes. C. EBV lytic transcripts are upregulated in miR-142 mutant cells. Four EBV lytic genes were measured by qRT-PCR in MutuI-iCas9 cells transduced with either a control gRNA (Neg3) or the top two scoring guide-RNAs (gRNAs) against miR-142 (10024 or 10033). Cells were either mock or anti-IgM treated for 48 hrs. Values are normalized to GAPDH and shown relative to anti- IgM treated Neg3 control cells. Shown is the average of six independent experiments. By Student’s t-test, *p<0.05. D. miR-142-3p and miR-142-5p expression in multiple B cell lines. Total RNA was extracted from DLBCLs (IBL1, IBL4, BCKN1), LCLs (LCL16.1, 17.1, 17.2), EBV-positive BL (KemI, RaeI, Raji, MutuI, Akata), and EBV-negative BL (BJAB, BL41, Ramos). miRNAs were assessed by Taqman qRT-PCR. Values are normalized to miR-16 and reported relative to IBL1.

Article Snippet: Inducible Cas9 vector pCW-Cas9-blast and gRNA expression vector pLentiGuide-puro were purchased from Addgene.

Techniques: CRISPR, Mutagenesis, Quantitative RT-PCR, Transduction, Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI 3K pathway inhibitors in PTEN ‐null triple negative breast cancer

doi: 10.15252/emmm.202011987

Figure Lengend Snippet: A Epitopes regulated by AZD8186 treatment in MDA‐MB‐468 cells. Representation of the results from RPPA analysis performed with 300 antibodies: comparison of the lysates from MDA‐MB‐468 treated with vehicle or AZD8186 250 nM for 2 (left) or 28 h (right). The dot‐plot shows the log 2 fold change in signal between vehicle and treated conditions vs the P ‐value (single sample t ‐test for non‐zero coefficient in the regression model) of the difference between the two conditions (biological triplicates) for each antibody. B Inducible expression of flagged‐Cas9 in MDA‐MB‐468. Cells transduced with the doxycycline‐inducible Cas9 (iCas9) construct were selected with hygromycin and single‐cell cloned. The MDA‐MB‐468 iCas9 clone was then treated with vehicle (ethanol) or doxycycline (DOX) for the indicated times and cell lysates probed with the indicated antibodies. Another cell line previously tested for inducible expression of Cas9 was used as a control (Ctrl), and predicted molecular weight of Cas9 is marked on the blot by an arrow. C Frequency and types of genetic modifications induced by a single sgRNA construct in MDA‐MB‐468 iCas9 clone. The cells were transduced with a lentiviral construct codifying for an sgRNA that targets the gene H1F0, whose inactivation is known to do not impact cell proliferation. The target genetic locus was sequenced and the frequency of the genetic modifications reported in the bar graph. D MDA‐MB‐468 parental and iCas9 clone showed similar sensitivity to PI3K pathway inhibitors. MDA‐MB‐468 parental cells or iCas9 clone was treated with serial dilutions of the indicated drugs for 4 days. Mean ± SD of triplicates and representative of two independent experiments. E–G KO efficiency in MDA‐MB‐468 iCas9 clone. Cells were transduced with a mix of five lentiviral constructs codifying for not overlapping non‐target sgRNAs (sgCTRL) or five sgRNAs designed to target GNB2 (E), EGFR (F), or ULK1 (G). Transduced cells were treated with doxycycline for the indicated times or for 8 days where not stated and cell lysates were probed with antibodies against the protein codified by the targeted gene or loading control. Quantification of the bands was performed by ImageLite software. H Inactivation of the essential gene PLK1 in MDA‐MB‐468 was used here as a killing control to validate the system. MDA‐MB‐468 iCas9 clone was transduced with a mix of five lentiviral constructs encoding control sgRNAs or sgRNAs against PLK1, and cell viability was measured by cell titer blue after 4 days of treatment with doxycycline. Mean ± SD of two independent experiments. I Scatter plot of raw phospho‐S6 signals from CRISPR‐Cas9 screening. Color corresponds to individual genes knocked‐out by sgRNAs, and each dot is associated with an individual measurement from a biological and technical replicate. Technical replicates are highlighted by different shapes and positioned in separate columns within each treatment condition. Solid black line indicates the mean level across all observations per drug treatment, and dashed horizontal black lines indicate standard deviation of the same. The horizontal cyan line indicates the threshold for lower outliers. Overlap between outliers can be observed between replicates and treatment conditions J Results of CRISPR‐Cas9 screening in combination with MK2206. The dot‐plots show for each gene knocked‐out by sgRNAs the fold change (log 2 ) between vehicle‐ and MK2206‐treated condition in the fluorescence signal (anti‐phosphoS6 Immunofluorescence) vs the P ‐value of the difference calculated by two‐sided t ‐test. The plots represent means of biological triplicates. Genes for which it was calculated a P < 0.0001 and Log 2 (fold change) > 0.25 are reported and highlighted in green; genes having a 0.0001 < P < 0.05 and a Log 2 (fold change) > 0.25 are reported and shown in red. K MDA‐MB‐468 iCas9 cells transduced with a mix of four GNB2 sgRNAs were single‐cell cloned after Cas9 induction and screened by immune‐fluorescence (IF) with an anti‐GNB2 antibody. GNB2‐expressing MDA‐MB‐468 cells were used as a positive control (+ ctrl), and three independent clones GNB2‐negative were identified (Clones 1–3). Signal from GNB2 was detected through Alexa Fluor‐488 conjugate secondary antibody (green), while nucleus staining was performed by DAPI (blue signal).

Article Snippet: Anti‐EGFR1 (from Francis Crick Institute Cell Service) was used in the Immuno‐precipitation experiments (5 μg antibody/1 mg protein). pLenti_BSD_sgRNA plasmid was a generous gift of Paola Scaffidi, and it was generated through replacement of GFP cassette with BSD cassette into the pLenti‐sgRNA‐Lib from Wei lab (Addgene Plasmid #53121). pCW‐Cas9 vector generated in David Sabatini's lab was obtained from Addgene (#50661).

Techniques: Expressing, Transduction, Construct, Clone Assay, Molecular Weight, Software, CRISPR, Standard Deviation, Fluorescence, Immunofluorescence, Positive Control, Staining

Journal: EMBO Molecular Medicine

Article Title: Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI 3K pathway inhibitors in PTEN ‐null triple negative breast cancer

doi: 10.15252/emmm.202011987

Figure Lengend Snippet: A, B Results of CRISPR‐Cas9 screening in combination with AZD8186 100 nM (A) or GDC0941 400 nM (B). The dot‐plots show for each gene knocked‐out by sgRNAs the fold change (log 2 ) between treated conditions (AZD8186 or GDC0941, respectively) and vehicle in the fluorescence signal (anti‐phosphoS6 immunofluorescence) vs the P ‐value of the difference calculated by two‐sided t ‐test. The plots represent means of biological triplicates. Genes for which it was calculated a P < 0.0001 and Log 2 (fold change) > 0.25 are reported and highlighted in green; genes having a 0.0001 < P < 0.05 and a Log 2 (fold change) > 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). C Box and whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941‐treated conditions for MDA‐MB‐468 cells transduced with non‐target control, GNB2, or GNG5 sgRNAs ( N = 2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper, and lower extremes of the box or hinge being the third and first quartiles, respectively, and the whiskers extending to the most extreme data values within 1.5 times the inter‐quartile range. Statistical significance of unpaired t ‐test **** P < 0.0001. D Biochemical analysis of GNB2 KO cells. Cell lysates of MDA‐MB‐468 parental cells and three MDA‐MB‐468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software. Source data are available online for this figure.

Article Snippet: Anti‐EGFR1 (from Francis Crick Institute Cell Service) was used in the Immuno‐precipitation experiments (5 μg antibody/1 mg protein). pLenti_BSD_sgRNA plasmid was a generous gift of Paola Scaffidi, and it was generated through replacement of GFP cassette with BSD cassette into the pLenti‐sgRNA‐Lib from Wei lab (Addgene Plasmid #53121). pCW‐Cas9 vector generated in David Sabatini's lab was obtained from Addgene (#50661).

Techniques: CRISPR, Fluorescence, Immunofluorescence, Positive Control, Whisker Assay, Transduction, Clone Assay, Software