Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: PC12 cells were pretreated with vehicle (0.02% DMSO), the Pyk2 inhibitor PF-431396 (3 μM), and the Src inhibitor PP2 (10 μM) or its inactive analogue PP3 (10 μM) for 5 min before application of bradykinin (Brad., 2 μM) or PMA (2 μM) for 10 min, extraction with 1% SDS at 65°C to ensure dissociation of all proteins, neutralization of SDS with excess of Triton X-100, and ultracentrifugation. Supernatants were analysed by direct IB with the indicated Pyk2 and Src antibodies. Some samples underwent IP with the anti-phosphotyrosine antibody 4G10 before IB with anti-Pyk2 antibody (top panel in A and quantification in B). IgG indicates control IP with non-immune mouse IgG. (A) Upper panel: total pY levels of Pyk2 determined by IP with 4G10 and IB with anti-Pyk2. Middle panel: pY402 levels of Pyk2 detected with anti-pY402 in corresponding lysates. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (B) Ratios of total pY of Pyk2 after 4G10 IP to total Pyk2 in lysates, normalized to control. F 5,63 =12.73. DMSO vs. Brad., P =0.012; DMSO vs. PMA, P <0.0001; Brad. vs PF-431396+Brad., P <0.0001; PMA vs. PF-431396+PMA, P =0.0001. (C) Ratios of pY402 to total Pyk2 signals in lysates, normalized to control. F 5,35 =10.94. DMSO vs. Brad., P =0.039; DMSO vs. PMA, P =0.0052; Brad. vs PF-431396+Brad., P =0.0005; PMA vs. PF-431396+PMA, P <0.0001. (D) Upper panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (E) Ratios of pY579 to total Pyk2 signals in lysates, normalized to control. F 5,36 =10.18. DMSO vs. Brad., P =0.0072; DMSO vs. PMA, P =0.021; Brad. vs PF-431396+Brad., P =0.0008; PMA vs. PF-431396+PMA, P =0.0011. (F,H) Upper panels: pY416 levels of Src detected with anti-pY416. Lower panels: Levels of total Src detected with anti-Src in same lysates. (G,I) Ratios of pY416 to total Src signals in lysates, normalized to control. (G) F 5,72 =4.464. DMSO vs. Brad., P =0.0167; DMSO vs. PMA, P =0.0226. (I) F 5,65 =11.06. DMSO vs. PMA, P =0.001; PMA vs. PP2+PMA, P <0.0001; DMSO vs. PP3+PMA, P =0.0042; PP3 vs. PP3+PMA, P =0.0086. (J) Upper panel: pY402 levels of Pyk2 detected with anti-pY402. Middle panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (K,L) Ratios of pY402 and pY579 to total Pyk2 signals in lysates, normalized to control. (K) F 5,42 =35.85. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PP3 vs. PP3+PMA, P =0.0001; DMSO vs. PP3+PMA, P =0.0068; DMSO vs. PP2+PMA, P =0.0001; DMSO vs. PP2, P <0.0001. (L) F 5,68 =13.40. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PP3 vs. PP3+PMA, P =0.0362; DMSO vs. PP3+PMA, P =0.0202. (B,C,E,G,I,K,L) Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis was by ANOVA with post-hoc Bonferroni’s multiple comparisons test; * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. Bradykinin and PMA induced phosphorylation of Pyk2 on Y402 and Y579 and of Src on Y416, all of which were blocked by PF-431396 and PP2 but not the inactive PP3.

Article Snippet: Rat pheochromocytoma cell line PC12 (ATCC Cat# CRL-1721; RRID:CVCL_0481, male) was grown in RPMI 1640 media (Gibco Cat#11875-101) containing 10% horse serum (HS, Gemini Bio Products Cat#100-508) and 5% FBS.

Techniques: Extraction, Neutralization, Control, Phospho-proteomics

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: PC12 cells were treated as in for analysis of tyrosine phosphorylation by IP with 4G10 and IB with anti-α 1 1.2. IgG indicates control IP with non-immune mouse IgG. The SU6656 was applied 5 min before PMA when indicated (SU, 10 μM). (A,C) Upper panels: pY of α 1 1.2 determined by 4G10 IP and α 1 1.2 IB. Lower panels: levels of total α 1 1.2 detected with anti-α 1 1.2 in corresponding lysates. (B,D) Ratios of pY signals in 4G10 IPs by IB with anti-α 1 1.2 to α 1 1.2 signals in lysates, normalized to control. Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis was by ANOVA with post-hoc Bonferroni’s multiple comparisons test. (B) F 5,50 =10.65. DMSO vs. Brad., P =0.0021; DMSO vs. PMA, P =0.0036; Brad. vs. PF-431396+Brad., P =0.0003; PMA vs. PF- 431396+PMA, P <0.0001. (D) F 7,31 =23.67. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PMA vs. SU+PMA, P <0.0001. (** P ≤0.01, *** P ≤0.001, **** P ≤0.0001). Bradykinin- and PMA-induced α 1 1.2 tyrosine phosphorylation was blocked by PF-431396, SU6656 and PP2 but not the inactive PP3.

Article Snippet: Rat pheochromocytoma cell line PC12 (ATCC Cat# CRL-1721; RRID:CVCL_0481, male) was grown in RPMI 1640 media (Gibco Cat#11875-101) containing 10% horse serum (HS, Gemini Bio Products Cat#100-508) and 5% FBS.

Techniques: Phospho-proteomics, Control

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: (A) Lysates from HEK293T/17 cells transfected with vectors encoding rat Pyk2 ( rPyk2-GFP) and either the Pyk2-targeting FIV lentivirus-derived, pVETL-Sh1-GFP (pFV-Pyk2-Sh1) or control (empty) pVETL-GFP (pFV-GFP) expression vectors, were immunoblotted (IB) with indicated antibodies. (B,C) IB analysis of indicated proteins in PC12 cultures incubated with viral particles containing pFV-Sh1-GFP (Sh1) FIV-based expression vector used in A or medium vehicle alone for 72h prior to treatment with either PMA (B), bradykinin (Brad.; C) or vehicle alone (-; B,C). Upper blots in B&C show anti-α 1 1.2 IBs of 4G10-anti-phosphotyrosine (pY) IP while middle and lower blots show direct IBs of indicated protein levels in input lysates. (D) Statistical analysis of the relative pY α 1 1.2 levels. F 5,41 =8.276. NT vs. PMA, P =0.0031; NT vs. Brad., P =0.0017; PMA vs. Sh1+PMA, P =0.001; Brad vs. Sh1+Brad, P =0.0433. (E,F) Direct IB analysis of indicated proteins in lysates of PC12 cultures transduced with HIV vector-derived lentiviral particles (e.g., pGFP-Pyk2-ShB-Lenti) containing expression cassettes for GFP and either the Pyk2-targeting (denoted pHV-Pyk-ShB and -ShC), Src-targeting (denoted pHV-Src-ShC and -ShD) or scrambled hairpin control (Cont.) shRNAs. In some cases (right blot) cultures were treated with PMA (+) or vehicle alone (-) before harvesting for IB. (G,H) IB analysis of indicated proteins from PC12 cultures infected with lentiviral particles containing HIV-GFP expression vectors as in E&F prior to treatment with either PMA (+) or vehicle (-). Upper panels show anti -α 1 1.2 IBs of 4G10-anti-pY IP while lower blots show direct IBs of input lysates with indicated antibodies. (I) Statistical analysis of relative α 1 1.2 pY levels. F 11,129 =6.180. NT vs. PMA, P <0.0001; PMA vs. Pyk2-ShB, P =0.0005; PMA vs. Pyk2-ShB+PMA, P =0.0029; PMA vs. Pyk2-ShC, P <0.0001; PMA vs. Pyk2-ShC+PMA, P =0.0002; PMA vs. Cont.-Sh, P =0.0019; PMA vs. Cont.-Sh+PMA, P >0.9999; PMA vs. Src-ShB, P =0.0007; PMA vs. Src-ShB+PMA, P <0.0001; PMA vs. Src-ShC, P <0.0001; PMA vs. Src-ShC+PMA, P< 0.0001. The bar graphs in (D) and (I) show ratios of quantified anti-α 1 1.2 IB signals in 4G10 IPs relative to α 1 1.2 IB signals in total lysates, normalized to not treated (NT) control. Comparisons are made between samples treated with PMA (or bradykinin in D) and each of the other indicated conditions. Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis by ANOVA with post-hoc Bonferroni’s multiple comparisons test (ns= not significant vs. PMA , *P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001).

Article Snippet: Rat pheochromocytoma cell line PC12 (ATCC Cat# CRL-1721; RRID:CVCL_0481, male) was grown in RPMI 1640 media (Gibco Cat#11875-101) containing 10% horse serum (HS, Gemini Bio Products Cat#100-508) and 5% FBS.

Techniques: Transfection, Derivative Assay, Control, Expressing, Incubation, Plasmid Preparation, Transduction, Infection

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 1. Schematic of experimental workflow for PC12 cell differentiation

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques: Cell Differentiation

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 2. Critical steps for aliquoting reagents and plating PC12 cells for differentiation (A) Set-up for aliquoting NGF, laminin, and CultureOne in tissue culture hood. Use one or two ice buckets and keep reagent vials and all tubes on ice while aliquoting. (B) Wells to avoid using in a 96-well plate (red line) due to increased potential for evaporation in these wells. (C) Improper (left) and proper (right) technique for plating PC12 cells for differentiation. Cell culture plates should be flat on the hood surface and the pipette should be held vertical when adding cells. Graphics in A and B were created with BioRender.com.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques: Evaporation, Cell Culture, Transferring

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 3. Different PC12 cell clonal variants vary in their differentiation rates Neurite densities of 4 different PC12 cell clonal variants each containing a different inducible mutant form of the androgen receptor (not expressed in these experiments), were quantified daily during differentiation until the culture reached a neurite density of 1,500 mm/mm2. The time it took for the 4 clonal variants to reach the target neurite density varied from 3–6 days of differentiation, and clonal variants also differed in their propensity to proliferate and clump during differentiation. Representative images of each clonal variant are shown on the day they reached the target neurite density. Three wells per clonal variant were analyzed and data represent mean G SD with a < 0.05. Images are cropped for clarity.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques: Mutagenesis, Variant Assay

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 4. AraC treatment reduces proliferation and cell clumping in differentiated PC12 cells PC12 cells were differentiated to a neurite density of 1,500 mm/mm2 and treated with 1 mM AraC for 48 h. Three days after AraC treatment, neurite-bearing cells exist as single cells in culture while proliferating cells are greatly reduced. (right, + AraC). In contrast, at the same timepoint in the absence of AraC treatment, neurite-bearing cells exist in clumps (left, - AraC, top) or are overtaken by proliferating cells (left, - AraC, bottom). Images are cropped for clarity.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques:

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 5. Cell confluency over 6 days of differentiation for western blot analysis Images depicting the confluency of PC12 cells every 2 days of differentiation, with corresponding lysis buffer volume used for cell lysis and average protein concentration in cell lysates. Images are cropped for clarity.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques: Western Blot, Lysis, Protein Concentration

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 6. NGF-induced neurite outgrowth of PC12 cells over 6 days of differentiation Upon NGF treatment, PC12 cells undergo a morphology change from a circular to triangular-shaped cell body and gradually extend neurites that develop bulbous terminal ends (arrows). Images are cropped for clarity.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques:

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 7. Differentiated PC12 cells express the neuronal markers Synapsin-1, b-III-Tubulin, and GAP43 (A) Expression of neuronal proteins Synapsin-1, b-III-Tubulin, and GAP43 increase over 6 days of differentiation, corresponding to neurite outgrowth. Phase-contrast images are cropped for clarity. (B) At 6 days of differentiation, neuronal proteins are visualized through immunofluorescence (b-III-Tubulin = red; Synapsin-1 and GAP43 = cyan (pseudo-colored)). Proteins are localized in the nucleus (Synapsin-1 and GAP43), cytoplasm (all), and neurites (all). Arrows indicate expression of Synapsin-1 and GAP43 in the bulbous terminal ends of the neurites. Images are cropped for clarity.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques: Expressing

Journal: STAR protocols

Article Title: Differentiating PC12 cells to evaluate neurite densities through live-cell imaging.

doi: 10.1016/j.xpro.2022.101993

Figure Lengend Snippet: Figure 9. Poor and proper dispersion of PC12 cells in a well An example of poor (left) and proper (right) dispersion of PC12 cells after plating. Cells should be plated as single cells and evenly distributed throughout the well. Images are cropped for clarity.

Article Snippet: Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.

Techniques: Dispersion