mab6495  (R&D Systems)

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Schematic of the protocol used to generate pulmonary neuroendocrine cells (PNECs) by stepwise differentiation of human pluripotent stem cells (hPSCs) to form: definitive endoderm (DE), day 3; anterior foregut endoderm (AFE), day 6; and lung progenitor cells (LPs) days 15–25. LPs were further differentiated into the types of lung cells (LCs) found in mature human lung parenchyma and airway epithelium, days 25–55. DAPT (10 μM) encourages the formation of PNECs, and addition of doxycycline (1 μM; DOX) induces expression of shRNAs against RB1 and TP53 mRNAs, as well as expression of MYC or MYC (T58A), as described in the text. ( B ) Western blot of extracts of RUES2 LCs at day 25 of differentiation protocol treated with DOX (1 μM for 72 hr); cells unexposed to DOX served as negative expression controls. Apparent differences in MYC protein levels may be attributable to the HA-tagged version of MYC (T58A), which migrates slightly slower than wild-type MYC protein. ( C ) Schematic representation of tumorigenesis experiments comparing injection sites (renal capsule or subcutaneous), DOX treatment (+/-DOX diet), and genotypes (see Materials and methods for additional details). Total numbers of animals are six to seven per experimental arm with two injection sites per mouse (right and left flank). Renal capsule injections were performed on a single kidney. Transgenic lines of RUES2 hESCs were differentiated and grown in DAPT (10 μM) from days 25 to 55. At day 55, PNECs were separated from other LCs by sorting for PE+ CGRP-expressing cells (see Materials and methods). PNECs were then injected either subcutaneously or into the renal capsular space in NOG mice, half of which then received DOX in their feed as described in Materials and methods. ( D ) Table summary of experiments with xenografted mice, indicating the number of animals that developed visible tumors (≥250 mm 3 in volume) at the site of injection or the number of visible metastases in the liver or lung. *, p<0.05; **, p<0.01 by Fisher’s test to denote significant differences between mice that did not receive DOX diet. As before, abbreviations for cell lines are: RP = shRB1+shTP53; RPM = shRB1+shTP53+WT MYC; RPM (T58A)=shRB1+shTP53+MYC (T58A). Figure 1—source data 1. Raw data of western blot bands in . Figure 1—source data 2. Raw data of .

Article Snippet: The lentiviral vectors expressing TET-inducible shRNAs against human RB1 construct (‘pSLIK sh human Rb 1534 hyg’ was a gift from Julien Sage; plasmid # 31500) , TET-inducible WT MYC (FUW-tetO-hMYC was a gift from Rudolf Jaenisch; plasmid # 20723) or mutant MYC (T58A) tagged at the N-terminus with three copies of a hemagglutinin tag (3X-HA) (pLV-tetO-myc T58A was a gift from Konrad Hochedlinger; plasmid # 19763) were obtained from Addgene and sequence-verified prior to use.

Techniques: Expressing, Western Blot, Injection, Transgenic Assay

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Subcutaneous xenografts formed with human embryonic stem cell (hESC)-derived pulmonary neuroendocrine cells (PNECs) with RP, RPM, or RPM (T58A) genotypes. Photographs of representative tumors formed with cells of the indicated genotypes are shown for RP and RPM; indicated scale of 1 cm. ( B ) Quantification of the tumor sizes and paired comparisons for a secondary in vivo experiment; n=5 animals per arm with single subcutaneous engraftments; **p<0.01. ( C ) H&E staining of the indicated tumors from panel B. ( D ) Gross and histologic pathology of renal capsular xenografts and liver metastases formed with the RPM or RPM (T58A) cells. Left panels, gross appearance of representative tumors within the liver (metastasis) or kidney (primary); right panels, H&E staining of primary and metastatic tumors ( T ) formed in kidney ( K ) and liver ( L ) in the RPM (left) or RPM (T58A) model. ( E ) Immunostaining of tumor samples from D. Samples of primary peri-renal tumors and hepatic metastases in mice injected with hESC-derived PNECs programmed to reduce levels of TP53 and RB1 mRNA and to express wild-type MYC were stained with antisera for Ki67 (left) or MYC (right). ( F ) Immunofluorescence staining for neuroendocrine markers ASCL1, NEUROD1, and CD56 from sections in E of the RPM tumors. ( G ) Immunofluorescence staining for neuroendocrine markers, CGRP, ASCL1, NEUROD1, and CD56 in the RP tumors. H&E staining serves as the bright-field comparison of the indicated tumors. Figure 3—source data 1. High resolution source data of .

Article Snippet: The lentiviral vectors expressing TET-inducible shRNAs against human RB1 construct (‘pSLIK sh human Rb 1534 hyg’ was a gift from Julien Sage; plasmid # 31500) , TET-inducible WT MYC (FUW-tetO-hMYC was a gift from Rudolf Jaenisch; plasmid # 20723) or mutant MYC (T58A) tagged at the N-terminus with three copies of a hemagglutinin tag (3X-HA) (pLV-tetO-myc T58A was a gift from Konrad Hochedlinger; plasmid # 19763) were obtained from Addgene and sequence-verified prior to use.

Techniques: Derivative Assay, In Vivo, Staining, Immunostaining, Injection, Immunofluorescence, Comparison

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence