Supplemental Fig. S1. The schematic diagrams of the expression constructs used in this study.

Supplemental data

Supplemental Fig. S1. The schematic diagrams of the expression constructs used in this study.

Supplemental Fig. S2. Ingenuity Pathway Analysis (IPA) of the 56 putative caspase substrates identified in STS-treated neurons. (A) Functional network with the second highest score (score 38, focus molecule 17). (B) Functional network with the third highest score (score 22, focus molecule 11). Colored nodes indicate putative substrates used in the pathway analysis.

Supplemental Fig. S3. In vitro caspase-3 cleavage assay for caspase-3 cleavage-deficient mutants. The V5-tagged wild-type and mutants of Gap43, Dbn1, and BASP1 were expressed in vitro using the TNT Quick Coupled Transcription/Translation System. Synthesized proteins were

then incubated with recombinant active caspase-3 (50 ng for Gap43 and BASP1; 10 ng for Dbn1) at 37 ºC and collected at various time points as indicated for immunoblotting against V5.

The reaction, with the caspase-inhibitor Z-VAD-FMK added, was incubated for 24 hrs. Numbers below the blots show the ratio of full-length protein density at each time point to that at the 0 time point.

Supplemental Fig. S4. Cleavage of MARCKSL1, BASP1 and Dbn1 by caspase-3 is not required for LTD induction. Cultured hippocampal slices (DIV 6-7) were biolistically transfected with a construct expressing the visual marker Venus along with an empty vector, or constructs expressing wild-type or mutant caspase-3 substrates (MARCKSL1, BASP1 or Dbn1). At 2-3 days after transfection, EPSCs were recorded in CA1 neurons before and after LTD induction by low-frequency stimulation of the Schaffer collateral pathway. (A) LTD is intact in cells transfected with constructs expressing wild-type or mutant MARCKSL1. (B) Wild-type and mutant BASP1 have no effect on LTD. (C) LTD is unchanged in cells transfected with wild-type

1

or mutant Dbn1. For each condition, the EPSC amplitude, which was normalized to baseline prior to LTD induction, was plotted as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis. n= 9-10 transfected neurons for each group.

Supplemental Fig. S5. (A) Basal synaptic transmission is not changed by overexpressing Gap43. Cultured hippocampal slices were biolistically transfected with the Gap43 construct or a control plasmid. EPSCs were recorded from transfected CA1 neurons by stimulating the Schaffer collateral pathway with stimulations at indicated intensities. Input-Output curve was plotted as the amplitude of eight consecutive EPSCs at 0.05 Hz versus the intensity of test stimuli. n = 11 neurons for the vector and 10 neurons for the Gap43-WT transfected groups. p= 0.46, 0.49, 0.49 and 0.58 (Gap43-WT vs. vector control) at 0.025mA, 0.05mA, 0.075mA and 0.1mA, respectively. (B) LTP is unchanged in cells transfected with Gap43 (D23/84/90/167A). n= 6 transfected neurons for each group. p= 0.86 (Gap43 quadruple mutant vs. vector control). For each condition, the EPSC amplitude was normalized to baseline prior to induction. Data was plotted as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis.

Supplemental Fig. S6. Caspase-3 cleavage sites identified in Gap43. Bold red indicates P1 Asp (D) in the caspase-3 cleavage sites identified. Bold black indicates the basic effector domain (a.a.

32-52) of Gap43 that binds acidic phospholipids, calmodulin, actin filaments, and PKC.

Supplemental Fig. S7. Basal AMPA receptor endocytosis is not affected by Gap43 mutants.

Cultured hippocampal neurons were transfected with constructs expressing wild-type or mutant Gap43 along with a plasmid expressing β-galactosidase, and analyzed for basal AMPA receptor

endocytosis (taking place without NMDA treatment) at 3 days after transfection. (A) Representative images of transfected neurons. The last row shows merged images of internalized

2

(green) and surface-remaining (red) GluA2. (B) Quantification for transfected neurons in (A).

The internalization index (integrated fluorescence intensity of internalized GluA2 / integrated fluorescence intensity of internalized GluA2 plus surface GluA2) was normalized to control cells transfected with the empty vector. n = 15 neurons for each group. Two-tailed Student’s t-test was used for statistical analysis. The graph shows mean ± SEM. Scale bar: 20 μm.

Supplemental Fig. S8. Images of live hippocampal neurons (DIV17~18) transfected with Gap43-mCherry or Gap43 (D23/84/90/167A)-mCherry along with EGFP-GluA2. Scale bar: 5 μm. The white arrows indicate axons labeled solely by Gap43-mCherry or Gap43 (D23/84/90/167A)-mCherry.

Supplemental Table S1. The complete list of identified unique SY-tagged peptides following an Asp residue in aligned proteins (P1 Asp cleaved peptides).

Supplemental Table S2. Exported peptide report from Scaffold for SY-tagged P1 Asp cleaved peptides.

Supplemental Table S3. The effect of mutant caspase-3 substrates on LTD.

Supplemental Table S4. Caspase-3 cleavage sites in Gap43 identified by three different methods in this study.

3

EF-1α ORF cDNA V5 Term

pEF-ENTR B

A. Mammalian expression vectors

B. E. coli expression vectors

T7 GB1 NusA Strep 8xHis TEV Gap43 ORF cDNA Term

pET-30a-GNSHT -Gap43

pCMV-ENTR B-EGFP-GluA2 CMV _peptide ^Signal EGFP GluA2 ORF cDNA V5 Term

a.a. 1- 25 a.a. 26- end

CMV Gap43 ORF cDNA mCherry Term

pCMV-ENTR B-Gap43-mCherry

Gap43 wt and mutants Dbn1 wt and mutant BASP1 wt and mutant MARCKSL1 wt

wt and D23/84/90/167A mutant

Supplemental Fig. S1

T7

4

Supplemental Fig. S2

A B

Cellular Assembly and Organization, Cellular Development, Embryonic Development

Cellular Assembly and Organization, Cell-To-Cell Signaling and Interaction, Cellular Function and Maintenance

5

Supplemental Fig. S3

Supplemental Fig. S4

Supplemental Fig. S5

8

Supplemental Fig. S6

9

Supplemental Fig. S7

Gap43-mCherry

Gap43 (D23/84/90/167A) -mCherry

EGFP-GluA2 Merge

Supplemental Fig. S8

11

Normalized EPSCs at 30 min after LTD induction

Cell number

p (vs. control, p < 0.05 considered significant)

Control (empty vector) 50 2% 9

BASP1 (WT) 53 2% 9 0.30

BASP1 (D135/159/166A) 56 2% 9 0.07

Dbn1 (WT) 54 2% 9 0.21

Dbn1 (D529A) 63 6% 10 0.05

MARCKSL1 (WT) 57 2% 9 0.07

MARCKSL1 (D63A) 56 2% 9 0.08

Gap43 (WT) 48 3% 9 0.70

Gap43 (D84A) 58 2% 9 0.03

Gap43 (D167A) 62 4% 9 0.01

Gap43 (D84/167A) 63 3% 9 0.007

Gap43 (D23/84/90/167A) 77 6% 10 0.001

Supplemental Table S3

12

Supplemental Table S4

13